Antibody to gdf8 and uses thereof

ABSTRACT

The disclosure provides novel molecules related to growth and differentiation factor-8 (GDF8), in particular epitopes specific to GDF8 and other specific antagonists of GDF8 in particular anti-GDF8 antibodies or antigen binding protein or fragment thereof which may inhibit GDF8 activity and signal in vitro and/or in vivo. The disclosure also provides for an immunoassay used to detect and quantitate GDF8. The disclosure also provides methods for diagnosing, preventing, ameliorating, and treating GDF8-associated disorders, e.g., degenerative orders of muscle, bone, and insulin metabolism. Finally, the disclosure provides pharmaceuticals for the treatment of such disorders by using the antibodies, polypeptides, polynucleotides, and vectors of the invention.

FIELD OF THE INVENTION

The technical field of the invention relates to the epitope(s) specificto growth and differentiation factor-8 (GDF8) and antagonists thereto(e.g., peptide mimetics, anti-GDF8 antibodies (e.g., mouse, human andhumanized antibodies, fragments thereof, etc.), recombinantpolynucleotides, inhibitory polynucleotides, etc.) that may be used toinhibit GDF8 activity in vitro and/or in vivoThe field further relatesto immunoassay methods for the detection of GF8 in biological . samplesas well as methods of treating, ameliorating, preventing, diagnosing,prognosing, and/or monitoring GDF8-associated disorders (e.g., muscledisorders, neuromuscular disorders, bone-degenerative disorders,metabolic or induced bone disorders, adipose disorders, glucosemetabolism disorders or insulin-related disorders), particularly inwomen of childbearing potential.

BACKGROUND OF THE INVENTION

Growth and differentiation factor-8 (GDF8), also known as myostatin, isa secreted protein and member of the transforming growth factor-beta(TGF-β) superfamily of structurally related growth factors. Members ofthis superfamily possess growth-regulatory and morphogenetic properties(Kingsley et al. (1994) Genes Dev. 8:133-46; Hoodless et al. (1998)Curr. Topics Microbiol. Immunol. 228:235-72). Human GDF8 is synthesizedas a 375 amino acid precursor protein that forms a homodimer complex.During processing, the amino-terminal propeptide, known as the“latency-associated peptide” (LAP), is cleaved and may remainnoncovalently bound to the homodimer, forming an inactive complexdesignated the “small latent complex” (Miyazono et al. (1988) J. Biol.Chem. 263:6407-15; Wakefield et al. (1988) J. Biol. Chem. 263:7646-54;Brown et al. (1999) Growth Factors 3:35-43; Thies et al. (2001) GrowthFactors 18:251-59; Gentry et al. (1990) Biochemistry 29: 6851-57;Derynck et al. (1995) Nature 316:701-05; Massague (1990) Ann. Rev. CellBiol. 12:597-641). Proteins such as follistatin and follistatin-relatedproteins including GASP-1 (Gamer et. al. (1999) Dev Biol. 208:222-232,US Patent Pub No. 2003-0180306-A1; US Patent Pub No. 2003-0162714-A1)and bind mature GDF8 homodimers and inhibit GDF8 biological activity.

An alignment of the deduced GDF8 amino acid sequence from variousspecies demonstrates that GDF8 is highly conserved (McPherron et al.(1997) Proc. Natl. Acad. Sci. U.S.A. 94:12457-61). The sequences ofhuman, mouse, rat, porcine, and chicken GDF8 are 100% identical in theC-terminal region, while baboon, bovine, and ovine GDF8 differ by a mere3 amino acids at the C-terminus. The high degree of GDF8 conservationacross species suggests that GDF8 has an essential physiologicalfunction.

GDF8 has been shown to play a major role in the regulation of muscledevelopment and homeostasis by inhibiting both proliferation anddifferentiation of myoblasts and satellite cells (Lee and McPherron(1999) Curr. Opin. Genet. Dev. 9:604-7; McCroskery et al. (2003) J.Cell. Biol. 162:1135-47). It is expressed early in developing skeletalmuscle, and continues to be expressed in adult skeletal muscle,preferentially in fast twitch types. GDF8 has also been implicated inthe production of muscle-specific enzymes (e.g., creatine kinase) andmyoblast proliferation (WO 00/43781).

Overexpression of GDF8 in adult mice results in significant muscle loss(Zimmers et al. (2002) Science 296:1486-88). Similarly, various studiesindicate that increased GDF8 expression is associated with HIV-inducedmuscle wasting (Gonzalez-Cadavid et al. (1998) Proc. Natl. Acad. Sci.U.S.A. 95:14938-43). In contrast, GDF8 knockout transgenic mice arecharacterized by a marked hypertrophy and hyperplasia of the skeletalmuscle and altered cortical bone structure (McPherron et al. (1997)Nature 387:83-90; Hamrick et al. (2000) Bone 27:343-49). Also, naturalmutations that render the GDF8 gene inactive have been shown to causeboth hypertrophy and hyperplasia in both animals and humans (Lee andMcPherron (1997), supra). For example, increases in skeletal muscle massare evident in natural GDF8 mutations in cattle (Ashmore et al. (1974)Growth 38:501-07; Swatland et al. (1994) J. Anim. Sci. 38:752-57;McPherron et al., supra; Kambadur et al. (1997) Genome Res. 7:910-15).

A number of human and animal muscle and bone disorders are associatedwith functionally impaired muscle tissue, and thus, may also beassociated with GDF8. For example, GDF8 may be involved in thepathogenesis of amyotrophic lateral sclerosis (“ALS”), musculardystrophy (“MD”; including Duchenne's muscular dystrophy, fascioscapularmuscular dystrophy, and facioscapulohumeral muscular dystrophy), muscleatrophy, carpal tunnel syndrome, organ atrophy, frailty, congestiveobstructive pulmonary disease (COPD), sarcopenia, cachexia, and musclewasting syndromes caused by other diseases and conditions.

GDF8 is also believed to participate in numerous other physiologicalprocesses and related disorders, including glucose homeostasis duringtype 2 diabetes development, impaired glucose tolerance, metabolicsyndromes (i.e., syndromes (e.g., syndrome X) involving the simultaneousoccurrence of a group of health conditions (which may include insulinresistance, abdominal obesity, dyslipidemia, hypertension, chronicinflammation, a prothrombotic state, etc.) that places a person at highrisk for type 2 diabetes and/or heart disease), insulin resistance(e.g., resistance induced by trauma such as burns or nitrogenimbalance), and adipose tissue disorders (e.g., obesity, dyslipidemia,nonalcoholic fatty liver disease, etc.) (Kim et al. (2000) Biochem.Biophys. Res. Comm. 281:902-06). Currently, few reliable or effectivetherapies exist to treat these disorders. The pathology of theseprocesses indicates GDF8 as a potential target in the treatment of theserelated disorders.

In addition to neuromuscular disorders in humans, there are also growthfactor-related conditions associated with a loss of bone, such asosteoporosis and osteoarthritis, which predominantly affect the elderlyand/or postmenopausal women. Such metabolic bone diseases and disordersinclude low bone mass due to chronic glucocorticoid therapy, prematuregonadal failure, androgen suppression, vitamin D deficiency, secondaryhyperparathyroidism, nutritional deficiencies, and anorexia nervosa.Although many current therapies for these conditions function byinhibiting bone resorption, a therapy that promotes bone formation wouldbe a useful alternative treatment. Because GDF8 plays a role in bonedevelopment as well as muscular development, GDF8 is also an excellentpharmacological target for the treatment of bone-degenerative disorders.

Like other members of the transforming growth factor-β (TGF-β) family,GDF8 is synthesized as a 376 amino acid precursor protein containing asignal sequence, a N-terminal propeptide domain, and a C-terminal domainconsidered as the active molecule. GDF8 is secreted in a latent form bybinding to it's propeptide (latency-associated peptide, LAP);proteolytic processing between the propeptide domain and the C-terminaldomain produces an N-terminal propeptide and the mature form of GDF8.Both unprocessed and mature GDF8 form disulfide-linked dimers, and theprocessed GDF8 dimer represents the only active form of the protein. Inserum, as well as in skeletal muscle, GDF8 can be found bound to severalproteins that are able to modulate its activation, secretion or receptorbinding.

GDF8 exerts its effects through a transmembrane serine/threonine kinaseheterotetramer receptor family, activation of which enhances receptortransphosphorylation, leading to the stimulation of serine/threoninkinase activity. It has been shown that the GDF8 pathway involves anactive GDF8 dimer binding to the high affinity receptor, ActIIRB, whichthen recruits and activates the transphosphorylation of the low affinityreceptor, ALK4/ALK5. It has also been shown that the proteins Smad 2 andSmad 3 are subsequently activated and form complexes with Smad 4 and arethen translocated to the nucleus, which then activate target genetranscription. Lee and McPherron (Proc Natl Acad Sci USA 2001, 98:9306-9311) have demonstrated that the ActRIIB receptor was able tomediate the influence of GDF8 in vivo, as expression of a dominantnegative form of ActIIRB in mice that mimics GDF8 gene knockout.

It has been shown that under the influence of GDF8, C2C12 myoblastsaccumulate in the G0/G1 and G2 phases of the cell-cycle, consequentlydecreasing the number of S-phase cells. Also, GDF8 induces failure ofmyoblast differentiation, associated with a strong decrease in theexpression of differentiation markers. GDF8 expression also decreasesthe apoptotic rate of cells under both proliferation and differentiationconditions (Thomas et al., J. Biol Chem 2000, 275:40235-40243).

Inhibition of myostatin (GDF8) expression leads to both musclehypertrophy and hyperplasia (Lee and McPherron, supra; McPherron et al.,supra). Myostatin negatively regulates muscle regeneration after injury,and lack of myostatin in GDF8 null mice results in accelerated muscleregeneration (McCroskery et al., (2005) J. Cell. Sci. 118:3531-41).Human anti-GDF8 antibodies (U.S. Published Application No. 2004/0142382)have been shown to bind GDF8 and inhibit GDF8 activity in vitro and invivo, including GDF8 activity associated with negative regulation ofskeletal muscle mass and bone density. For example,myostatin-neutralizing antibodies increase body weight, skeletal musclemass, and muscle size and strength in the skeletal muscle of wild typemice (Whittemore et al. (2003) Biochem. Biophys. Res. Commun.300:965-71) and the mdx mouse, a model for muscular dystrophy(Bogdanovich et al. (2002) Nature 420:418-21; Wagner et al. (2002) Ann.Neurol. 52:832-36). Furthermore, myostatin antibodies in these micedecrease the damage to the diaphragm, a muscle that is also targetedduring ALS pathogenesis. It has been hypothesized that the action ofgrowth factors, such as HGF, on muscle may be due to inhibition ofmyostatin expression (McCroskery et al. (2005), supra), thereby helpingto shift the balance between regeneration and degeneration in a positivedirection. However, these prior art antibodies were not specific forGDF8, i.e., these antibodies have high affinity for other members of theTGF-β superfamily, such as BMP11.

To date, all known inhibitors of GDF8 activity (e.g., propeptide,soluble ActRIIB receptor, anti-GDF8 antibodies, etc.) also neutralizethe biological activities of other factors (e.g., BMP11, activin, etc.)that have important biological functions. For example, activin and BMP11play important roles during embryogenesis. Activin βA is identified as acritical gonadal growth factor, and BMP11 is responsible for homeotictransformation of the axial skeleton. Homozygous BMP11 knockout mice areperinatal lethal; mice with one wild type copy of the BMP11 gene areviable but have skeletal defects. Since activin and BMP11 play importantroles during embryogenesis, an antagonist that inhibits GDF8 and otherfactors, e.g., BMP11 poses theoretical safety risks that could presenteither as toxicity in treated patients or as reproductive toxicity in,e.g., women of childbearing potential. Thus, there is a need forcompounds and methods of treatment that contribute to an overallincrease in muscle mass and/or strength and/or bone density,particularly in humans, but do not interfere with, e.g., BMP11. In otherwords, there is a need for specific inhibition of GDF8 activity intreatments of GDF8-associated disorders for which it is desirable toincrease muscle mass, size, strength, etc., particularly in women withchildbearing potential.

As methods of using GDF-8 modulating agents are developed, there is aneed to develop methods to monitor and to optimize the administration ofsuch agents to an individual. In particular, the ability to measureGDF-8 protein levels in biological fluids has important implications forongoing clinical trials. For example, circulating GDF-8 levels might bediagnostic for pathological conditions that could benefit fromanti-GDF-8 therapy, or might predict which individuals are more likelyto respond to anti-GDF-8 therapy. In addition, changes in GDF-8 levelsin peripheral blood during anti-GDF-8 treatment may be an earlyindicator of later measurable response in muscle mass and/or function.

In order to accomplish such optimization goals, methods to detect ormonitor GDF-8 protein levels in biological fluids, such as serum andplasma are needed. It is desirable to monitor GDF-8 levels prior to,during, and post treatment with a GDF-8 modulating agent in order toidentify appropriate individuals for such treatment, monitor responsesto the treatment, and follow post-treatment progress, for example. Inparticular, methods allowing the detection and/or quantitation ofendogenous GDF-8 levels in response to administration of GDF-8modulating agents, including GDF-8 inhibitors and anti-GDF-8 antibodiesare needed.

It is accordingly a primary object of the present invention to providecompounds and methods that specifically inhibit GDF8 activity as well asimmunological assays to detect and quantitate GDF-8 levels in biologicalsamples, such as, for example, in serum and plasma.

SUMMARY OF THE INVENTION

The invention is based on the discovery of antibodies or antigen bindingproteins that specifically bind to Growth and Differentiation Factor 8(GDF8) that specifically antagonize at least one GDF8 activity (e.g.,GDF8 binding to its receptor or other GDF8-mediated signaling events).The present invention is also based on the identification of theepitopes on GDF8 recognized by these specific anti-GDF8 antibodies orantigen binding proteins, since the antibodies of the invention arespecific to GDF8 and do not bind specifically to, for example, BMP11.

In addition to providing epitope(s) specific to GDF8, the invention alsoprovides antagonists specific for GDF8 (also referred to herein as“specific GDF8 antagonists,” “GDF8 antagonist,” and the like), e.g.,antagonists that specifically antagonize (e.g., inhibit, reduce, and/orneutralize) at least one GDF8 activity (e.g., GDF8-mediated signalingevents (e.g., GDF8 binding to its receptor (e.g., its ALK4/ALK5receptor)), and do not significantly antagonize BMP11 activity. Thepresent invention also provides methods for detecting and quantifyingGDF-8 in biological samples. In certain embodiments, the methodscomprise immunoassays, and the sample is serum and/or plasma. Theinvention further provides kits for use in the methods of the invention.The invention also provides methods of using the disclosed specific GDF8antagonists in methods of treating (which includes ameliorating,preventing, diagnosing, prognosing) or monitoring GDF8-associateddisorders, e.g., muscle disorders, neuromuscular disorders,bone-degenerative disorders, metabolic or induced bone disorders,adipose disorders, glucose metabolism disorders, insulin-relateddisorders, etc.

Thus, in one aspect, the invention provides antagonists to GDF8 whereinthe antagonists comprises at least one of a peptide mimetic of a GDF8binding domain; an isolated nucleic acid that encodes an amino acid fora peptide mimetic of a GDF8 binding domain; an inhibitory polynucleotidespecific to GDF8; and an anti-GDF8 antibody or antigen binding proteinthat specifically binds to GDF8 and does not specifically bind to BMP11.

In one embodiment, the invention provides the antagonist describedherein wherein the antagonist is a peptide mimetic of a GDF8 bindingdomain and is selected from the group consists essentially of an aminoacid sequence selected from the group consisting of the amino acidsequence of SEQ ID NO:4; the amino acid sequence of SEQ ID NO:6; theamino acid sequence of SEQ ID NO:8; the amino acid sequence of SEQ IDNO:10; and the amino acid sequence of SEQ ID NO:12. In some embodiments,the invention provides an antagonist that is a peptide mimetic asdescribed herein and is cyclized. In some embodiments, the inventionprovides an antagonist that is a peptide mimetic as described herein andis cyclized by means of a disulfide bond. In any one or more embodimentsthe invention provides an antagonist that is a peptide mimetic asdescribed herein that has at least one D-amino acid. In some embodimentsthe invention provides an antagonist that is a peptide mimetic that maybe used as an immunogen.

In another embodiment the invention provides an antagonist describedherein wherein the antagonist is an anti-GDF8 antibody, antigen bindingprotein or fragment thereof that specifically binds to GDF8 but does notspecifically bind to BMP11, wherein the antibody or antigen bindingprotein is selected from the group consisting of: polyclonal antibody; amonoclonal antibody; a monospecific antibody; polyspecific antibody;humanized antibody; a tetrameric antibody; a tetravalent antibody; amultispecific antibody; a single chain antibody; a domain-specificantibody; a single domain antibody; a domain-deleted antibody; a fusionprotein; an ScFc fusion protein; a single-chain antibody; chimericantibody; synthetic antibody; recombinant antibody; hybrid antibody;mutated antibody; CDR-grafted antibodies; an antibody fragment which mayinclude an Fab; an F(ab′)2; an Fab′ fragment; an Fv fragment; asingle-chain Fv (ScFv) fragment; an Fd fragment; a dAb fragment; anantigen binding protein which may include diabodies; a CDR3 peptide; aconstrained FR3-CDR3-FR4 peptide; a nanobody; a bivalent nanobody; smallmodular immunopharmaceuticals (SMIPs); a shark variable IgNAR domain;and a minibody. In some embodiments the antagonist of the invention is amonoclonal antibody. In some embodiments, the antagonist of theinvention is a humanized antibody.

In some embodiments the invention provides an antagonist that is anantibody, antigen binding protein or fragment thereof that is specificfor GDF8 that is comprised of at least one complementarity determiningregions (CDR) comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO:19, the amino acidsequence of SEQ ID NO:20, the amino acid sequence of SEQ ID NO:21, theamino acid sequence of SEQ ID NO:22, the amino acid sequence of SEQ IDNO:23, the amino acid sequence of SEQ ID NO:24, the amino acid sequenceof SEQ ID NO:25, the amino acid sequence of SEQ ID NO:26, the amino acidsequence of SEQ ID NO:27, the amino acid sequence of SEQ ID NO:28, theamino acid sequence of SEQ ID NO:29, the amino acid sequence of SEQ IDNO:30.

In some embodiments the antagonist of the invention is an anti-GDF8antibody, antigen binding protein or fragment thereof that comprises aheavy chain which comprises a first, second and third CDR, wherein thefirst CDR comprise an amino acid selected from the amino acid sequenceof SEQ ID NO:19; and the amino acid sequence of SEQ ID NO:25, the secondCDR comprises an amino acid selected from sequence of SEQ ID NO:20; andthe amino acid sequence of SEQ ID NO:26 and the third CDR comprises anamino acid selected from the amino acid sequence of SEQ ID NO:21; andthe amino acid sequence of SEQ ID NO:27. In some embodiments theantibody or antigen binding protein of the invention comprises a heavychain which comprises an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO:14 and the aminoacid sequence of SEQ ID NO:18.

In some embodiments, the antagonist of the invention is an anti-GDF8antibody or antigen binding protein that comprises a light chain whichcomprises a first, second and third CDR, wherein the first CDR comprisesan amino acid selected from the amino acid sequence of SEQ ID NO:22; andthe amino acid sequence of SEQ ID NO:28, the second CDR comprises anamino acid selected from the amino acid sequence of SEQ ID NO:23; andthe amino acid sequence of SEQ ID NO:29, the third CDR comprising anamino acid selected from the amino acid sequence SEQ ID NO:24; and theamino acid sequence of SEQ ID NO:30. In some embodiments the antagonistof the invention is an anti-GDF8 antibody or antigen binding proteinwhich comprises a light chain comprising an amino acid sequence selectedfrom the group consisting of: the amino acid sequence of SEQ ID NO:16;and the amino acid sequence of SEQ ID NO:17.

In some embodiments the antagonist of the invention is an anti-GDF8antibody or antigen binding protein that comprises a light chaincomprising the amino acid sequence of SEQ ID NO:16, and furthercomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:14. In some embodiments the antagonist of the invention is ananti-GDF8 antibody or antigen binding protein that comprises a lightchain comprising the amino acid sequence of SEQ ID NO:17, and furthercomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:18. In some embodiments, the invention provides a polynucleotide thatencodes any one or more of the amino acids comprising the GDF8antagonist of the invention, as described herein.

In some embodiments the antagonist of the invention is an inhibitorypolynucleotide that specifically binds to GDF8 and is selected from thegroup consisting of: an siRNA molecule and an antisense molecule. Insome embodiments the invention provides any one or more of thepolynucleotides described herein that encode the antagonists of theinvention. In some embodiments the invention provides a polynucleotidethat encodes an amino acid sequence selected from the group consistingof the amino acid sequence of SEQ ID NO:4; the amino acid sequence ofSEQ ID NO:6; the amino acid sequence of SEQ ID NO:8; the amino acidsequence of SEQ ID NO:10; and the amino acid sequence of SEQ ID NO:12.In another embodiment the invention provides a polynucleotide whereinthe isolated polynucleotide consists essentially of a nucleic acidsequence selected from the group consisting of: the nucleic acidsequence of SEQ ID NO:3, the nucleic acid sequence of SEQ ID NO:5, thenucleic acid sequence of SEQ ID NO:7, the nucleic acid sequence of SEQID NO:9, the nucleic acid sequence of SEQ ID NO:11, and the nucleic acidsequences of fragments thereof.

In some embodiments the invention provides a host cell comprising anyone or more polynucleotides of the invention, wherein the polynucleotideis operably linked to a regulatory sequence. In another embodiment theinvention provides a vector comprising any of the polynucleotides of theinvention. In another embodiment the invention provides a host cellcomprising a vector comprising any one or more of the polynucleotides ofthe invention.

In some embodiments the invention provides a method for producing a GDF8antagonist from a cultured a host cell as described herein comprisingany one or more of the polynucleotides of the invention and isolatingthe GDF8 antagonist expressed by the host cell. In yet anotherembodiment the invention provides an isolated GDF8 antagonist producedby the method for producing a GDF8 antagonist as described herein.

In another aspect of the invention, the invention provides an assay todetect the presence of GDF8 in a sample from a subject which comprisesthe following steps: combining (i) the sample with (ii) a capturereagent that specifically binds GDF8 and (iii) a detection reagent thatspecifically binds GDF8 and detecting whether or not specific bindingoccurs between the capture reagent and GDF8 wherein detection ofspecific binding indicates the presence of GDF8 in the sample.

In one embodiment of the invention the GDF8 in the sample is dissociatedfrom the GDF8 binding proteins and anti-GDF8 present in the sample. Inone embodiment, the assay of the invention further comprises combiningthe sample with an acidic buffer prior to the combination of the samplewith the capture reagent, as described herein. In another embodiment theacidic buffer of the assay of the invention has a pH between about pH1.0 and pH 6.0. In another embodiment the pH of the acidic buffer isabout pH2.5.

In one embodiment, the invention provides any one or more of the assaysdescribed herein wherein the sample is selected from the groupconsisting of serum, whole blood, plasma, biopsy sample, tissue sample,cell suspension, saliva, oral fluid, cerebrospinal fluid, amnioticfluid, milk, colostrums, mammary gland secretion, lymph, urine, sweat,lacrimal fluid, gastric fluid, synovial fluid and mucus. In anotherembodiment, the invention provides that the sample is chosen from wholeblood, serum or plasma.

In one embodiment, the invention provides any one or more of the assaysdescribed herein wherein the detecting step comprises at least one of asandwich assay and a competitive binding assay. In some embodiments thedetecting step comprises a sandwich assay In some embodiments, thedetecting step comprises at least one of: detecting a change inrefractive index at a solid optical surface in contact with the sample;detecting a change in luminescence; measuring a change in color;detecting a change in radioactivity; measuring using biolayerinterferometry; measuring using cantilever-detection; measuring usinglabel-free intrinsic Imaging; and measuring using acoustic-detection. Insome embodiments, the detection step of the invention measures a changein color. In some embodiments, the detecting step comprises an assayselected from the group consisting of: an enzyme-linked immunosorbentassay (ELISA); an electro-chemiluminescent assay (ECL); radioimmunoassay(RIA); solid-phase radioimmunoassay (SPRIA); immunoblotting;immunoprecipitation; Fluorescent Activated Cell Sorting (FACS). Inanother embodiment the detecting step comprises and ELISA.

In one embodiment the presence of GDF8 is detected by the specificbinding of a compound to the detection reagent that specifically bindsGDF8 wherein the compound further comprises a detectable label. Inanother embodiment the detectable label comprises at least one labelselected from the group consisting of an enzyme label; a luminescentlabel, a protein label; a vitamin label; a chromophoric label; aradioisotopic label and an electron dense molecule label. In anotherembodiment the detectable label is a protein label and further comprisesbiotin.

In one embodiment, the assay of the invention provides a capture reagentthat is selected from the group consisting of an anti-GDF8 antibody,antigen binding protein or fragment thereof; a GDF8 binding protein; anda GDF8 binding domain. In another embodiment, the assay of the inventionprovides that the capture reagent is an anti-GDF8 antibody, antigenbinding protein or fragment thereof and is selected from the group ofconsisting of RK35, RK22, MYO-028, MYO-029 and JA16. In some embodimentsthe capture reagent is RK35. In some embodiments the capture reagent isRK22.

In one embodiment of the assay of the invention provides the detectionreagent is selected from the group consisting of: an anti-GDF8 antibody,antigen binding protein or fragment thereof; a GDF8 binding protein anda GDF8 binding domain. In one embodiment the detection reagent is ananti-GDF8 antibody, antigen binding protein or fragment thereof and isselected from the group consisting of: RK22 and RK35. In anotherembodiment, the assay of the invention provides the detection reagent isRK35. In another embodiment, the assay of the invention provides thedetection reagent is RK22.

In one embodiment the invention provides an assay wherein the capturereagent is RK22 and the detection reagent is RK35. In anotherembodiment, the invention provides an assay wherein the capture reagentis RK35 and the detection reagent is RK22.

In another aspect of the invention, the invention provides an assay toquantitate the presence of GDF8 in a sample from a subject whichcomprises the following steps: combining (i) the sample with (ii) acapture reagent that specifically binds GDF8 and (iii) a detectionreagent that specifically binds GDF8 detecting whether or not specificbinding occurs between the capture reagent and GDF8 and quantitate thelevel of GDF8 in the sample, wherein detection of specific bindingindicates the presence of GDF8 in the sample and can be quantitated. Inone embodiment of the invention provides the GDF8 in the sample isdissociated from the GDF8 binding proteins and anti-GDF8 present in thesample. In one embodiment, the assay of the invention further comprisescombining the sample with an acidic buffer prior to the combination ofthe sample with the capture reagent, as described herein. In anotherembodiment the acidic buffer of the assay of the invention has a pHbetween about pH 1.0 and pH 6.0. In another embodiment the pH of theacidic buffer is about pH2.5.

In another aspect of the invention, the invention provides apharmaceutical composition for treating (which includes ameliorating,and/or preventing) a GDF8-associated disorder in a subject comprising apharmaceutically acceptable carrier and at least one GDF8 antagonist isselected from the group consisting of a peptide mimetic of a GDF8binding domain; an isolated nucleic acid that encodes an amino acid fora peptide mimetic of a GDF8 binding domain; an inhibitory polynucleotidespecific to GDF8 and an anti-GDF8 antibody, antigen binding protein orfragment thereof that specifically binds to GDF8 and does notspecifically bind to BMP11.

In one embodiment the pharmaceutical composition of the inventioncomprises a peptide mimetic of a binding domain specific for GDF8consisting essentially of an amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO:4; the amino acidsequence of SEQ ID NO:6; the amino acid sequence of SEQ ID NO:8; theamino acid sequence of SEQ ID NO:10; and the amino acid sequence of SEQID NO:12.

In one embodiment the pharmaceutical composition of the inventioncomprises a isolated nucleic acid that encodes for an amino acidspecific to GDF8 consists essentially of a nucleic acid sequenceselected from the group consisting of the nucleic acid sequence of SEQID NO:3; the nucleic acid sequence of SEQ ID NO:5; the nucleic acidsequence of SEQ ID NO:7; the nucleic acid sequence of SEQ ID NO:9; thenucleic acid sequence of SEQ ID NO:11 and the nucleic acid sequences offragments thereof.

In one embodiment the pharmaceutical composition of the inventioncomprises an anti-GDF8 antibody, antigen binding protein or fragmentthereof that specifically binds with GDF8 and does not specifically bindBMP11 which comprises a light chain comprising the amino acid sequenceof SEQ ID NO: 16, and wherein the antibody further comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 14. In anotherembodiment, the pharmaceutical composition of the invention provides ananti-GDF8 antibody, antigen binding protein or fragment thereof thatspecifically binds with GDF8 and does not specifically bind BMP11comprises a light chain comprising the amino acid sequence of SEQ ID NO:18, and wherein the antibody, antigen binding protein or fragmentthereof further comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 17.

In one embodiment the pharmaceutical composition of the inventioncomprises an anti-GDF8 antibody or antigen binding protein thatspecifically binds with GDF8 and does not bind BMP11 and comprises atleast one complementarity determining region (CDR) comprising an aminoacid sequence selected from the group consisting of the amino acidsequence of SEQ ID NO: 19, the amino acid sequence of SEQ ID NO: 20, theamino acid sequence of SEQ ID NO:21, the amino acid sequence of SEQ IDNO:22, the amino acid sequence of SEQ ID NO:23, the amino acid sequenceof SEQ ID NO:24, the amino acid sequence of SEQ ID NO:25, the amino acidsequence of SEQ ID NO:26, the amino acid sequence of SEQ ID NO:27, theamino acid sequence of SEQ ID NO:28, the amino acid sequence of SEQ IDNO:29, the amino acid sequence of SEQ ID NO:30.

In one embodiment the pharmaceutical composition of the invention isused to treat a GDF8 associated disorder selected from the groupconsisting of a muscle disorder, neuromuscular disorder,bone-degenerative disorder, metabolic or induced bone disorder, adiposedisorder, glucose metabolism disorder, and insulin-related disorder in amammalian patient. In another embodiment, the pharmaceutical compositionof the invention wherein the GDF8 associated disorder is selected fromthe group consisting of muscular dystrophy, ALS, muscle atrophy, organatrophy, carpal tunnel syndrome, frailty, congestive obstructivepulmonary disease, sarcopenia, cachexia, muscle wasting syndromes,obesity, type-2 diabetes, impaired glucose tolerance, metabolicsyndromes, insulin resistance, nutritional disorders, premature gonadalfailure, androgen suppression, secondary hyperparathyroidism,osteoporosis, osteopenia, osteoarthritis, low bone mass, vitamin Ddeficiency, and anorexia nervosa.

Another aspect of the invention provides a method of treating (whichincludes ameliorating and/or preventing) a GDF8-associated disorder in amammalian patient comprising administering to the patient atherapeutically effective amount of an antagonist specific for GDF8 thathas little to no toxicity. In another embodiment the method of theinvention provides that the antagonist of the invention is selected fromthe group consisting of a peptide mimetic of a GDF8 binding domain; anisolated nucleic acid that encodes an amino acid for a peptide mimeticof a GDF8 binding domain; an inhibitory polynucleotide specific to GDF8and an anti-GDF8 antibody, antigen binding protein or fragment thereofthat specifically binds to GDF8 and does not specifically bind to BMP11.

In one embodiment the invention provides that the antagonist of theinvention is a peptide mimetic of a GDF8 binding domain and the GDF8binding domain consists essentially of an amino acid sequence selectedfrom the group consisting of the amino acid sequence of SEQ ID NO:4; theamino acid sequence of SEQ ID NO:6; the amino acid sequence of SEQ IDNO:8; the amino acid sequence of SEQ ID NO:10; and the amino acidsequence of SEQ ID NO:12.

In one embodiment the method of treatment of the invention provides amethod of treatment wherein the antagonist of the invention is aisolated nucleic acid that encodes for an amino acid specific to GDF8consists essentially of a nucleic acid sequence selected from the groupconsisting of the nucleic acid sequence of SEQ ID NO:3, the nucleic acidsequence of SEQ ID NO:5, the nucleic acid sequence of SEQ ID NO:7, thenucleic acid sequence of SEQ ID NO:9, the nucleic acid sequence of SEQID NO:11, and the nucleic acid sequences of fragments thereof.

In one embodiment of the invention provides a method of treatmentwherein the antagonist of the invention is an anti-GDF8 antibody,antigen binding protein or fragment thereof that specifically binds withGDF8 and does not specifically bind to BMP11 and comprises a light chaincomprising the amino acid sequence of SEQ ID NO:16, and furthercomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:14. In some embodiments the method of treatment of the inventionprovides that the antagonist of the invention is an anti-GDF8 antibody,antigen binding protein or fragment thereof that specifically binds withGDF8 and does not specifically bind to BMP11 which comprises a lightchain comprising the amino acid sequence of SEQ ID NO:18, and whereinthe antibody, antigen binding protein or fragment thereof furthercomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:17. In some embodiments the method of treatment of the inventionprovides that the antagonist of the invention is an anti-GDF8 antibody,antigen binding protein or fragment thereof that specifically binds withGDF8 and does not specifically bind to BMP11 that comprises at least onecomplementarity determining region (CDR) comprising an amino acidsequence selected from the group consisting of: the amino acid sequenceof SEQ ID NO:19, the amino acid sequence of SEQ ID NO:20, the amino acidsequence of SEQ ID NO:21, the amino acid sequence of SEQ ID NO:22, theamino acid sequence of SEQ ID NO:23, the amino acid sequence of SEQ IDNO:24, the amino acid sequence of SEQ ID NO:25, the amino acid sequenceof SEQ ID NO:26, the amino acid sequence of SEQ ID NO:27, the amino acidsequence of SEQ ID NO:28, the amino acid sequence of SEQ ID NO:29, theamino acid sequence of SEQ ID NO:30.

In one embodiment of the invention the method of treatment of theinvention provides that the GDF8-associated disorder is selected fromthe group consisting of a muscle disorder, neuromuscular disorder,bone-degenerative disorder, metabolic or induced bone disorder, adiposedisorder, glucose metabolism disorder, and insulin-related disorder in asubject. In some embodiments of the invention the method of treatment ofthe invention provides that the GDF8-associated disorder is selectedfrom the group consisting of muscular dystrophy, ALS, muscle atrophy,organ atrophy, carpal tunnel syndrome, frailty, congestive obstructivepulmonary disease, sarcopenia, cachexia, muscle wasting syndromes,obesity, type-2 diabetes, impaired glucose tolerance, metabolicsyndromes, insulin resistance, nutritional disorders, premature gonadalfailure, androgen suppression, secondary hyperparathyroidism,osteoporosis, osteopenia, osteoarthritis, low bone mass, vitamin Ddeficiency, and anorexia nervosa.

Another aspect of the invention provides a method of diagnosing,prognosing or detecting whether a subject is afflicted with aGDF8-associated disorder comprising the steps of: obtaining a firstsample from the subject; combining a first sample with the antagonist ofthe invention; detecting the presence of GDF8 in the first sample;quantitating the level of GDF8 in the first sample; obtaining a secondsample from a subject not afflicted with the GDF8-associated disorder;combining the second sample with the antagonist; detecting the level ofGDF8 in the second sample; quantitating the level of GDF8 in the secondsample and comparing the levels of GDF8 in the first and second samples,wherein an increase, decrease, or similarity in the level of GDF8 in thesecond sample compared to the first sample indicates whether theGDF8-associated disorder has changed in severity.

Another aspect of the invention provides a method of monitoring theseverity of a GDF8-associated disorder comprising the steps of: (i)obtaining a first sample from the subject; (ii) combining a first samplewith the antagonist as in any one of claims 1-16; (iii) detecting thepresence of GDF8 in the first sample; (iv) quantitating the level ofGDF8 in the first sample; (v) obtaining a second sample from a subjectnot afflicted with the GDF8-associated disorder; (vi) combining thesecond sample with the antagonist; (vii) detecting the level of GDF8 inthe second sample; (viii) quantitating the level of GDF9 in the secondsample and (ix) comparing the levels of GDF8 in the first and secondsamples, wherein an increase, decrease, or similarity in the level ofGDF8 in the first sample compared to the second sample indicates whetherthe GDF8-associated disorder has changed in severity.

Another aspect of the invention provides a method of prognosing thelikelihood that a subject will develop a GDF8-associated disordercomprising the steps of: (i) obtaining a first sample from the subject;(ii) combining a first sample with the antagonist as in any one ofclaims 1-16; (iii) detecting the presence of GDF8 in the first sample;(iv) quantitating the level of GDF8 in the first sample; (v) obtaining asecond sample from a subject not afflicted with the GDF8-associateddisorder; (vi) combining the second sample with the antagonist; (vii)detecting the level of GDF8 in the second sample; (viii) quantitatingthe level of GDF9 in the second sample and (ix) comparing the levels ofGDF8 in the first and second samples, wherein an increase, decrease, orsimilarity in the level of GDF8 in the second sample compared to thefirst sample indicates the likelihood that the subject will develop aGDF8-associated disorder.

Another aspect of the invention provides a method of prognosing thelikelihood that a subject will develop a GDF8-associated disordercomprising the steps of: (i) obtaining a first sample from the subject;(ii) combining a first sample with the antagonist as in any one ofclaims 1-16; (iii) detecting the presence of GDF8 in the first sample;(iv) quantitating the level of GDF8 in the first sample; (v) obtaining asecond sample from a subject not afflicted with the GDF8-associateddisorder; (vi) combining the second sample with the antagonist; (vii)detecting the level of GDF8 in the second sample; (viii) quantitatingthe level of GDF9 in the second sample and (ix) comparing the levels ofGDF8 in the first and second samples, wherein an increase, decrease, orsimilarity in the level of GDF8 in first sample compared to the secondsample indicates the likelihood that the subject will develop aGDF8-associated disorder.

Another aspect of the invention provides a use of a pharmaceuticalcomposition in the preparation of a medicament for treating (whichincludes ameliorating, and/or preventing) a GDF8-associated disorder ina mammalian patient wherein the pharmaceutical composition comprises apharmaceutically acceptable carrier and at least one GDF8 antagonist isselected from the group consisting of a peptide mimetic of a GDF8binding domain; an isolated nucleic acid that encodes an amino acid fora peptide mimetic of a GDF8 binding domain; an inhibitory polynucleotidespecific to GDF8 and an anti-GDF8 antibody, antigen binding protein orfragment thereof that specifically binds to GDF8 and does notspecifically bind to BMP11.

Another aspect of the invention provides a kit for detecting thepresence of GDF8 in a sample from a subject, the kit comprising acapture reagent that specifically binds GDF8 and a detection reagentthat specifically binds GDF8 wherein detection of specific binding ofGDF8 to the capture and detection reagents indicate the presence of GDF8in the sample. In some embodiments the kit of the invention furthercomprises an acidic buffer.

Another aspect of the invention provides a kit for the quantitation ofGDF8 in a sample from a subject, the kit comprising a capture reagentthat specifically binds GDF8 and a detection reagent that specificallybinds GDF8 wherein detection of specific binding of GDF8 to the captureand detection reagents allow quantitation of GDF8 in the sample. In someembodiments the kit of the invention further comprises an acidic buffer.

In another aspect the invention provides an antibody as described hereinwherein the antibody is an anti-GDF8 antibody, antigen binding proteinor fragment thereof that specifically binds to GDF8 but does notspecifically bind to BMP11, wherein the antibody or antigen bindingprotein is selected from the group consisting of: polyclonal antibody; amonoclonal antibody; a monospecific antibody; polyspecific antibody;humanized antibody; a tetrameric antibody; a tetravalent antibody; amultispecific antibody; a single chain antibody; a domain-specificantibody; a single domain antibody; a domain-deleted antibody; a fusionprotein; an ScFc fusion protein; a single-chain antibody; chimericantibody; synthetic antibody; recombinant antibody; hybrid antibody;mutated antibody; CDR-grafted antibodies; an antibody fragment which mayinclude an Fab; an F(ab′)2; an Fab′ fragment; an Fv fragment; asingle-chain Fv (ScFv) fragment; an Fd fragment; a dAb fragment; anantigen binding protein which may include diabodies; a CDR3 peptide; aconstrained FR3-CDR3-FR4 peptide; a nanobody; a bivalent nanobody; smallmodular immunopharmaceuticals (SMIPs); a shark variable IgNAR domain;and a minibody. In some embodiments the antagonist of the invention is amonoclonal antibody. In some embodiments, the antagonist of theinvention is a humanized antibody. In some embodiments the antibody isan anti-GDF8 antibody. In some embodiments the antibody of the inventionis an anti-GDF8 antibody or antigen binding protein that comprises alight chain comprising the amino acid sequence of SEQ ID NO:16, andfurther comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:14. In some embodiments the antibody of the invention is ananti-GDF8 antibody or antigen binding protein that comprises a lightchain comprising the amino acid sequence of SEQ ID NO:17, and furthercomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:18.

BRIEF DESCRIPTION THE SEQUENCES

DNA and amino acid sequences are set forth in the Seq. Listing and areenumerated in Table 1.

TABLE 1 SEQ ID NO DESCRIPTION 1 A.A. seq. mature human GDF8 2 A.A. seq.human GDF8 precursor 3 DNA seq. peptide mimetic(GE1) 4 A.A. seq. peptidemimetic (GE1) 5 DNA seq. peptide mimetic (GE2) 6 A.A. seq. peptidemimetic (GE2) 7 DNA seq. peptide mimetic (GE3) 8 A.A. seq. peptidemimetic (GE3) 9 DNA seq. peptide mimetic (GE4) 10 A.A. seq. peptidemimetic (GE4) 11 DNA seq. peptide mimetic (GE5) 12 A.A. seq. peptidemimetic (GE5) 13 DNA seq. RK22 VH mouse 14 A.A. seq. RK22 VH mouse 15DNA seq. RK22 VL mouse 16 A.A. seq. RK22 VL mouse 17 A.A. seq. RK22 VLhumanized 18 A.A. seq. RK22 VH humanized 19 A.A. seq. CDR H1 (Kabat) 20A.A. seq. CDR H2 (Kabat) 21 A.A. seq. CDR H3 (Kabat) 22 A.A. seq. CDR L1(Kabat) 23 A.A. seq. CDR L2 (Kabat) 24 A.A. seq. CDR L3 (Kabat) 25 A.A.seq. CDR H1 (AbM) 26 A.A. seq. CDR H2 (AbM) 27 A.A. seq. CDR H3 (AbM) 28A.A. seq. CDR L1 (AbM) 29 A.A. seq. CDR L2 (AbM) 30 A.A. seq. CDR L3(AbM) 31 DNA seq. RK35 VH 32 DNA seq. RK35 VL 33 DNA seq. MYO-029 VH 34DNA seq. MYO-029 VL 35 DNA seq. ActRIIB 36 A.A. seq. ActRIIB

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B demonstrates the binding (O.D. 450 nm; y-axis) ofvarious concentrations (ng/ml; x-axis) of supernatant from RK22 and RK35(a control antibody that binds with both GDF8 and BMP11) expressinghybridomas to GDF8 or BMP11.

FIG. 2 shows the kinetic rate constants for the interaction between RK22antibody and GDF8 as determined by BIAcore 2000 system Sensor Chip SA.

FIG. 3 shows the induction of pGL3 (CAGA)12-TGF-β promoter reporter geneactivity as measured by luciferase activity (LCPS; y-axis) in A204rhabdosarcoma cells treated with 10 ng/ml of GDF8 in the absence (10mg/ml) or presence of various concentrations (M Ig; x-axis) of the RK22and RK35 antibody and other RK antibodies (A through E) that bind toeither GDF8 and/or BMP11.

FIG. 4 shows the weight (g; y-axes) of gastrocnemius (Gastroc),quadricep (Quad), and anterior tibialis (Tibialis anterior) musclesdissected from SCID mice after four weeks of treatment with vehicle inthe absence (vehicle), or presence of 1, 10 or 40 mg/kg/week of RK22 orMyo-29.

FIG. 5 shows binding to ActRIIB (OD450; y-axis) by GDF8 alone or GDF8preincubated with various concentrations ([M]; x-axis) of RK22,non-specific antibody, other RK antibodies (D and E), a control antibodythat blocks GDF8 binding to ActRIIB (RK35), control IgG antibody, orsoluble ActRIIB.

FIG. 6 shows the resulting of epitope mapping dot blots of 20 ng/ml of acontrol antibody, RK22 antibody, incubated with 48 individual andoverlapping 13-residue peptides representing the entire mature GDF8peptide. Under each dot blot is the sequence of GDF8 with the GDF8epitopes for the antibodies underlined (as indicated in the respectivedot blots).

FIG. 7 shows the alignment of the RK22 variable heavy chain domain(RK22_VH) with the human germline framework sequences of DP-7(DP-7_germl_VH) and DP-5 (DP-5_germl_VH); the amino acids of the murineRK22 variable heavy chain domain that are changed in the humanized RK22variable heavy chain domain are designated with an asterisk and the CDRsof RK22 are boxed and underlined.

FIG. 8 shows the alignment of the RK22 variable light chain domain withthe human germline framework sequence of DPK 24 VL; the amino acids ofthe murine RK22 variable light chain domain that are changed in thehumanized RK22 variable light chain domain are designated with anasterisk and the CDRs of RK22 are boxed and underlined.

FIG. 9A shows a comparison of immunoassay formats: RK35, the capturereagent and biotinylated RK22 as the detection reagent. FIG. 9B showsRK22 as the capture with biotinylated RK35 as the detection antibody.

FIG. 10. demonstrates that the ELISA assays previously described exhibitbackground, and that this background is likely a human anti-mouseantibody (HAMA) effect. Assay background was due to serum crossreactivity with monoclonal antibody RK22 that was used as a captureantibody in the ELISA. The same effect is observed when RK35 is used asthe capture antibody (data not shown).

FIG. 11 shows the results of a GDF-8 ELISA where the RK35 antibody wascoated on the plate, and the background of the ELISA was reduced usingthe commercially available reagent IIR (Immunoglobulin InhibitingReagent-Bioreclamation, NY). The results using IIR compare favorablywith background with buffer only.

FIG. 12 shows that the antibody RK35 does not bind to GDF-8 in thepresence of MYO-029. MYO-029 antibody was coated onto HBX assay platesand GDF-8 was added at 1200 pg/ml with increasing concentrations ofbiotinylated detection antibodies (RK22 or RK35). No signal is producedwith biotinylated RK35. The results indicate crossreactivity betweenRK35 and MYO-029 for binding to GDF-8.

FIGS. 13A and 13B show that MYO-029 can be used as an inhibitor of GDF-8in an ELISA assay. Increasing concentrations of MYO-029 with a constantconcentration of GDF-8 (250 pg/ml) spiked into assay buffer or into 10%human serum were assayed for GDF-8 via ELISA. FIG. 13A shows that thereis approximately 30% inhibition of signal when RK22 is used as thecapture antibody. FIG. 13B shows that inhibition is nearly 100% (from 5to 20 pg/ml of MYO-029) when RK35 is used as the capture antibody. Alsoshown is the reduction in background signal (serum) by the use of 2IR(also known as “IIR”). Total signal is shown in both graphs and has notbeen converted to percent inhibition.

FIGS. 14A and 14B show the results from a “spike recovery experiment,”where GDF-8 was added to 100% serum in three separate serum samples(Sera #1, #2, and #3). Each sample was analyzed +/−20 pg/ml MYO-029. Theaddition of 20 pg/ml MYO-029 blocks assay signal at all concentrationsof GDF-8 tested (FIG. 13A). FIG. 13B shows the results from aspike-recovery assay where sera, but no MYO-029, was added. The resultsshow a linear increase in signal with the addition of GDF-8.

FIG. 15 shows a comparison of standard curves generated in normal mouse,knockout (KO) mouse and human serum. The slope of the curve with THSTbuffer alone is much steeper than those generated in serum and cannot beused to quantitate values in serum.

FIG. 16 demonstrates observed versus expected GDF8 values as generatedin THST buffer.

FIG. 17 presents the results from an experiment where it is shown thatMYO-029 can be inactivated/dissociated from GDF-8 when heated toapproximately 80° C. Serum samples were spiked with GDF-8 or latentGDF-8 +/−5 pg/ml MYO-029 and heated to 80° C. The results show thatheating the MYO-029 samples restores the ability to detect GDF-8,indicating that MYO-029 is dissociated from GDF-8, and activated uponheating to 80° C. The results also show that when MYO-029 is added backto the heated sample, the GDF-8 signal is once again reduced. Also shownis an increase in endogenous GDF-8 signal upon heating that is even morepronounced in samples spiked with latent GDF-8 as compared to matureGDF-8 samples.

FIG. 18 shows the results of an ELISA assay for GDF-8 before and afterdepleting GDF-8 from the human serum. Normal human serum +/−20 pg/mlMYO-029 was analyzed for GDF-8 at room temperature (“RT”), and afterheating to 80° C. These values were compared to the same samples thatwere first depleted of GDF-8 by pre-heating to 65° C. for 10 minutes,and passing three times over a 1 mg MYO-029 affinity column. The resultsindicate that depleting GDF-8 from human serum is effective at reducingthe background level of GDF-8 in this ELISA. The results also show thatheating the depleted serum does not show the original increase in signalthat is observed with normal serum upon heating. This heated/GDF-8depleted (H/D) serum can be used for generation of GDF-8 standardcurves.

FIG. 19 shows a graph of standard curves that were generated in heated,GDF-8 depleted serum, and then spiked with increasing concentrations ofmature GDF-8 or latent GDF-8. The standard curves were assayed at roomtemperature (RT) or with heating to 80° C. Serum samples spiked withlatent GDF-8 show a large signal increase after heating that was notobserved in samples spiked with mature GDF-8.

FIG. 20 shows the analysis of two standard curves run on differentplates on two successive days. Heated/GDF-8 depleted serum with knownconcentrations of mature GDF-8 is used to generate the standard curves.OD values from each curve are plotted versus mature GDF-8 concentration.Non-linear regression curve fit is a more accurate correlation for GDF-8values generated in heated/GDF-8 depleted serum. The curve fit has abetter correlation co-efficient and has more range for accuracy thanlinear regression curves. Software from Prism Graph was used tocalculate GDF-8 concentration from the OD values generated in the GDF-8ELISA assay.

FIG. 21 shows the results of an assay to measure free and total GDF-8 innine normal serum samples. Results are displayed as +/−MYO-029. Valuesare given in pg/ml of GDF-8 and correspond to the endogenous level ofGDF-8 in 100% serum.

FIG. 22 shows the results of an assay to measure free and total GDF-8 ineight normal serum samples. The graph represents two separateexperiments conducted on two separate days. On the second day of theexperiment, total GDF-8 was analyzed by heating the samples for tenminutes (as compared to the five minute heating conducted on the firstday of the experiment). Values ranged from 227 to 1241 pg/ml for freeGDF-8 and from 514 to 4329 pg/ml for total GDF-8.

FIG. 23 shows the results of an assay to measure GDF-8 via ELISA afterheat denaturation. Aliquots of human serum were incubated at roomtemperature for one hour +/−10 μg/ml of MYO-029. The samples were nextheat denatured in a gradient thermocycler at various temperatures priorto ELISA analysis. Quantitation of GDF-8 levels in test samples wasperformed by interpolation from the assay results of a standard curveconsisting of a dilution series of purified recombinant GDF-8 dimer ofknown concentration spiked into pooled human serum depleted of GDF-8 byaffinity chromatography. Maximum detection of GDF-8 in the absence ofMYO-029 occurs at approximately 60° C. The presence of MYO-029 masksdetection of GDF-8 in serum at low temperature, but at temperaturesgreater than 65° C., GDF-8 detection was partially restored.

FIGS. 24A and 24B show that GDF-8 can be detected in the presence ofMYO-029 at low pH. In FIG. 24A, GDF-8 dimer diluted in THST buffer +/−10μg/ml of MYO-029 was diluted five-fold into either assay buffer atneutral pH (THST), 200 mM sodium acetate pH 5.0 (NaOAc), 200 mMphosphate-citrate buffer pH 3 or 7 (PO4Cit), or 200 mM glycine-HCl pH2.5 (Gly). Samples were then diluted 1:1 into ELISA wells (coated withRK35 antibody) containing either THST buffer or non-buffered Tris.Dilution of GDF-8 with solutions of different pH and bufferingcapacities and the subsequent dilution into THST or non-buffered Trisallowed measurement of the efficiency of the analyte capture step at apH range from approximately 3 to 8. Under assay conditions approachingneutral pH, MYO-029 reduced GDF-8 detection (THST/THST). GDF-8 acidifiedwith glycine-HCl and subsequently diluted into THST buffer maintained asufficiently low pH to prevent MYO-029 binding and allowed fulldetection of GDF-8 in the presence or absence of MYO-029 (Gly/THST).However, dilution of glycine-acidified GDF-8 into non-buffered Trisresulted in analyte capture conditions at pH greater than 7 and reduceddetection of GDF-8 in the presence of MYO-029 (Gly/Non-buffered Tris).FIG. 24B shows a schematic of the ELISA assay at pH3 (left panel) andpH7 (right panel).

FIG. 25 provides the results of an experiment that shows that the murineantibody RK35 binds to GDF-8 under acidic conditions, while MYO-029 doesnot. Human serum was pre-incubated +/−various concentrations of RK35 orMYO-029, and was diluted five-fold with THST buffer (neutral pH) orglycine-HCl (acidic pH). The serum was then added to ELISA wells thatcontained plate-bound RK35 and either THST or glycine-HCl buffer. In theabsence of RK35 or MYO-029, more endogenous GDF-8 is detected in acidicconditions than in neutral conditions due to complex dissociation andthe release of free GDF-8 proteins (see first two bars). The last sixbars represent data obtained at acidic pH. Increasing amounts of RK35 insolution was able to bind and compete for GDF-8 binding to theplate-bound RK35, resulting in decreased detection of GDF-8 under acidicconditions. MYO-029 was incapable of binding GDF-8 in solutionapproaching pH3, leaving the GDF-8 in solution available to bind to theRK35 antibody coated on the ELISA plate.

FIG. 26 shows that increasing the MYO-029 concentration to 100 μg/ml didnot diminish detection of GDF-8 using the acid dissociation ELISAprotocol. Human serum +/−10, 40, or 100 μg/ml of MYO-029 was assayedfollowing heat or acid dissociation. The concentration of GDF-8 wasestimated by interpolation from a standard curve of recombinant GDF-8mature dimer spiked into pooled human serum depleted of GDF-8. In theabsence of MYO-029, the concentration of GDF-8 in serum was determinedto be ˜1 ng/ml when analyte capture was performed at near neutral pH(HS). Detection of GDF-8 following the release of serum binding proteinsvia heat dissociation was ˜3 ng/ml in the absence of MYO-029 (HS+Myo,63° C.; no Ab). In the presence of MYO-029, even at the lowestconcentration tested, heat dissociation failed to permit the same levelof detection as in the absence of MYO-029 (HS+MYO, 73° C.). Acidtreatment of serum samples detected very similar amounts of GDF-8independent of the amount of MYO-029 present (HS+MYO, Acid).

FIG. 27 demonstrates that acidic conditions during analyte capture donot reduce assay specificity. Acidification of serum resulted in greatersignal detection (white bars vs. black bars) in all wild type (WT)animals tested, Cynomolgus monkey (NHP-31), mouse (WT Mm), and dairy cow(WT cow). Serum measured at neutral or acidic pH from either agenetically engineered GDF-8 knockout (KO Mm) mouse or the naturallyoccurring GDF-8 KO Belgian Blue cow (KO Cow) failed to produce a signalover the plate background.

FIG. 28A-C contrast three different methods of calibration curve fittingfor five GDF-8 ELISA plates in terms of their relative errors of theback-calculated concentrations for calibrating standards. Results fromfive GDF-8 ELISA plates are plotted. Relative error is defined as(B−N)/N×100, where B is the back-calculated concentration for a standardusing the calibration curve, and N is the nominal concentration of thestandard. Three curve fitting methods were: 1) FIG. 36A—4-parameterlogistic model on optical density by least squares (LS); 2) FIG.36B—4-parameter logistic model on square root of optical density by LS;and 3) FIG. 36C—5-parameter logistic model on square root of opticaldensity by LS. Reference lines are at −20 and 20.

FIG. 29A. Serum myostatin levels in normal and myostatin-null animals.Serum from Belgian Blue cattle, normal cattle (wt cow), myostatin nullmice (mstn KO mouse) and wild-type littermates (wt mouse) were measuredunder dissociative, acidic conditions (pH2.5), and values wereextrapolated from a standard curve of recombinant human myostatin inmyostatin-deficient serum matrix. Bars represent mean in +/−SD ofreplicate samples (n=3). Myostatin concentrations in myostatin-nullanimals fell below the concentration of the lowest calibrator sample(147 pg/mL).

FIGS. 29B and 29C. Myostatin levels in cynomolgous monkey serum measuredin the myostatin ELISA under non-dissociative (pH 8.0, panel B) ordissociative (pH 2.5, panel C) conditions following addition ofincreasing concentrations of anti-myostatin antibody MYO-029 or solublemyostatin receptor ActRIIB-Fc. Bars represent mean+/−SD of replicatesamples (n=3). Dashed line indicates the LLQ.

FIG. 30 A. Box and whisker plots of serum myostatin levels (mean, SD,median and first and third quartile values) in young and older men. Thehorizontal line in the box represents the mean, the lower and upperboundaries of the box represent the first and third quartiles, and thevertical bars represent the SDs. *, P=0.03. FIG. 30B. Regression plotshowing correlation of baseline myostatin levels with lean body mass inyoung men. FIG. 30 C. Regression plot showing correlation of baselinemyostatin levels with lean body mass in older men.

FIG. 31. Changes in myostatin levels in young men in response toadministration of graded doses of testosterone (bar diagram showingmean+/−SEM levels at baseline, and days 56 and 140. FIG. 31A shows themyostatin levels at baseline, treatment day 56, and 140 in young (leftpanel) and older men (right panel). The data are mean+/−SEM. *, P valueas in comparison to baseline levels. Myostatin levels on day 140 werenot significantly different from baseline levels. FIG. 31B shows thepercent change from baseline in serum myostatin levels from baseline today 56 in young and older men. *, P=0.03

FIG. 32. Regression plots showing correlation of the change in myostatinlevels from baseline to day 56 and changes in total and freetestosterone concentrations and lean body mass in young and older men.FIG. 32A shows the linear regression plot of percent change in myostatinlevels from baseline to day 56 and percent change in serum totaltestosterone concentrations in young men. FIG. 32B shows the linearregression plot of percent change in myostatin levels from baseline today 56 and percent change in serum total testosterone concentrations inolder men. FIG. 32C shows the linear regression of percent change inmyostatin levels from baseline to day 56 and percent change in serumfree testosterone concentrations in young men. FIG. 32D shows the linearregression of percent change in myostatin levels from baseline to day 56and percent change in serum free testosterone concentrations in oldermen. FIG. 32E shows the linear regression of percent change in myostatinlevels from baseline to day 56 and percent change in lean body mass frombaseline to day 140 in young men. FIG. 32F shows the linear regressionof percent change in myostatin levels from baseline to day 56 andpercent change in serum lean body mass from baseline to day 140 in oldermen.

FIG. 33. Box and whisker plots of myostatin levels (mean, SD, median andfirst and third quartile values) in young menstruating, surgicallymenopausal, and older women. The horizontal line in the box representsthe mean, the lower and upper boundaries of the box represent the firstand third quartiles, and the vertical bars represent the SDs. Myostatinlevels in the three groups were not statistically significant.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include reference to the plural unless the contextclearly dictates otherwise.

The term “antibody,” as used herein, refers to an immunoglobulin or afragment thereof, and includes, but is not limited to: a polyclonalantibody, a monoclonal antibody, a monospecific antibody, polyspecificantibody, humanized antibody, a tetrameric antibody, a tetravalentantibody, a multispecific antibody, a single chain antibody, adomain-specific antibody, a single domain antibody, a domain-deletedantibody, a fusion protein, an ScFc fusion protein, a single-chainantibody, chimeric antibody, synthetic antibody, recombinant antibody,hybrid antibody, mutated antibody, CDR-grafted antibodies and antibodyfragments which includes: Fab, F(ab′)2, an Fab′ fragment, an Fvfragment, a single-chain Fv (ScFv) fragment, an Fd fragment, and a dAbfragment or any chemically or genetically manipulated counterparts, ofthe foregoing that retains antigen binding function.

The invention also provides antigen binding proteins, which aredifferent from antibodies as described herein, which include diabodies,a CDR3 peptide, a constrained FR3-CDR3-FR4 peptide, a nanobody (USpatent application 2008/0107601), a bivalent nanobody, small modularimmunopharmaceuticals (SMIPs), a shark variable IgNAR domain (WO03/014161), a minibody and any fragment or chemically or geneticallymanipulated counterparts that retain antigen-binding function.Typically, such fragments would comprise an antigen-binding domain. Aswill be recognized by those of skill in the art, any of such molecules,e.g., a “humanized” antibody or antigen binding protein, may beengineered (for example “germlined”) to decrease its immunogenicity,increase its affinity, alter its specificity, or for other purposes.

Antibodies of the invention can be made, for example, via traditionalhybridoma techniques (Kohler et al., Nature 256:495-499 (1975)),recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage displaytechniques using antibody libraries (Clackson et al., Nature 352:624-628(1991); Marks et al., J. Mol. Biol. 222:581-597 (1991)). For variousother antibody production techniques, see Antibodies: A LaboratoryManual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. Theterm “antigen” refers to a compound, composition, or immunogenicsubstance that can stimulate the production of antibodies or a T-cellresponse, or both, in an animal, including compositions that areinjected or absorbed into an animal. The immune response may begenerated to the whole molecule, or to a portion of the molecule (e.g.,an epitope or hapten). The term may be used to refer to an individualmacromolecule or to a homogeneous or heterogeneous population ofantigenic macromolecules. An antigen reacts with the products ofspecific humoral and/or cellular immunity. The term “antigen” broadlyencompasses moieties including proteins, polypeptides, antigenic proteinfragments, nucleic acids, oligosaccharides, polysaccharides, organic orinorganic chemicals or compositions, and the like. The term “antigen”includes all related antigenic epitopes. Epitopes of a given antigen canbe identified using any number of epitope mapping techniques, well knownin the art. See, e.g., Epitope Mapping Protocols in Methods in MolecularBiology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J.For example, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, allincorporated herein by reference in their entireties. Similarly,conformational epitopes are readily identified by determining spatialconformation of amino acids such as by, e.g., x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols, supra. Furthermore, for purposes of the present invention, an“antigen” can also include includes modifications, such as deletions,additions and substitutions (generally conservative in nature, but theymay be non-conservative), to the native sequence, so long as the proteinmaintains the ability to elicit an immunological response. Thesemodifications may be deliberate, as through site-directed mutagenesis,or through particular synthetic procedures, or through a geneticengineering approach, or may be accidental, such as through mutations ofhosts, which produce the antigens. Furthermore, the antigen can bederived or obtained from any virus, bacterium, parasite, protozoan, orfungus, and can be a whole organism. Similarly, an oligonucleotide orpolynucleotide, which expresses an antigen, such as in nucleic acidimmunization applications, is also included in the definition. Syntheticantigens are also included, for example, polyepitopes, flankingepitopes, and other recombinant or synthetically derived antigens(Bergmann et al. (1993) Eur. J. Immunol. 23:2777 2781; Bergmann et al.(1996) J. Immunol. 157:3242 3249; Suhrbier, A. (1997) Immunol. and CellBiol. 75:402 408; Gardner et al. (1998) 12th World AIDS Conference,Geneva, Switzerland, Jun. 28 Jul. 3, 1998).

The term “antigen-binding domain,” “active fragments of an antibody”, orantigen binding protein or the like refers to the part of an antibody orantigen binding protein molecule that comprises the area specificallybinding to or complementary to a part or all of an antigen. Where anantigen is large, an antibody may only bind to a particular part of theantigen. The “epitope,” “active fragments of an epitope,” or “antigenicdeterminant” or the like is a portion of an antigen molecule that isresponsible for specific interactions with the antigen-binding domain ofan antibody. An antigen-binding domain may be provided by one or moreantibody variable domains (e.g., a so-called Fd antibody fragmentconsisting of a VH domain). An antigen-binding domain may comprise anantibody light chain variable domain (VL) and an antibody heavy chainvariable domain (VH) (U.S. Pat. No. 5,565,332).

A “sample” is biological material collected from cells, tissues, organs,or organisms, for example, to detect an analyte. Exemplary biologicalsamples include serum, blood, plasma, biopsy sample, tissue sample, cellsuspension, saliva, oral fluid, cerebrospinal fluid, amniotic fluid,milk, colostrum, mammary gland secretion, lymph, urine, sweat, lacrimalfluid, gastric fluid, synovial fluid, mucus, and other clinicalspecimens and samples.

The term “capture reagent” refers to a reagent, for example an antibodyor antigen binding protein, capable of binding a target molecule oranalyte to be detected in a biological sample. Typically, the capturereagent is immobilized, for example on an assay surface, for example, asolid substrate or reaction vessel. A “GDF-8 capture reagent”specifically binds to GDF-8.

A “detection reagent” is a reagent, for example an antibody or antigenbinding protein, that is used in the immunoassays of the presentinvention to specifically bind to a target protein, for example, GDF-8.A detection reagent may optionally comprise a detectable label. Adetection reagent typically recognizes and binds the target protein at abinding site or epitope distinct from that of the capture reagent. Thedetection reagent may be coupled to a detectable label. A “GDF-8detection reagent” specifically binds to GDF-8.

The term “complimentary determining region” or “CDR” refers to thehypervariable regions of an antibody or antigen binding proteinmolecule, consisting of three loops from the heavy chain and three fromthe light chain that together form the antigen binding domain.

The term “detecting” is used in the broadest sense to include bothqualitative and quantitative measurements of a target analyte, hereinmeasurements of a specific target molecule such as GDF-8 or BMP-11. Theassay methods described herein can be used to identify the presence ofGDF-8 or BMP-11 in a biological sample, or may be used to quantify anamount of GDF-8 or BMP-11 in a sample.

A “detection agent” or “detection reagent” may be used in the methods ofthe present invention to detect the signal generated from a detectionantibody or antigen binding protein that comprises an indirect label. Adetection agent or reagent is a protein or small molecule that allowsdetection of a GDF-8 modulating agent or a complex. In a preferredembodiment, the detection agent specifically binds to a GDF-8 modulatingagent. A detection agent may optionally comprise a detectable label. Adetection agent may also be itself detected by a substance comprising adetectable label. A GDF-8 modulating agents detected by the methodsprovided herein, may also be used in the methods to detect other GDF-8modulating agents, for example.

A “disorder associated with GDF8 activity”, “disorder associated withGDF8”, “GDF8-associated disorder,” or the like refers to a disorder thatmay be caused, in full or in part, by dysregulation of GDF8, (e.g.,abnormally increased or decreased expression and/or activity of GDF8)and/or a disorder that may be treated, ameliorated, prevented,prognosed, and/or monitored by regulating and/or monitoring GDF8 proteinand/or activity. GDF8 associated disorders include muscle disorders,neuromuscular disorders, bone-degenerative disorders, metabolic orinduced bone disorders, adipose disorders, glucose metabolism disorders,or insulin-related disorders.

The term “effective dose” “therapeutically effective dose” or “effectiveamount” refers to a dosage or level that is sufficient to ameliorateclinical symptoms of, or achieve a desired biological outcome (e.g.increasing muscle mass, muscle strength and/or bone density) inindividuals, including individuals having a GDF-8 associated disorder.Such amount would be sufficient to reduce the activity of GDF-8associated with negative regulation of skeletal muscle mass and bonedensity, for example. Therapeutic outcomes and clinical symptoms mayinclude increase in muscle mass, improved cardiovascular indicators orimproved glucose metabolism regulation. A GDF-8 inhibitor can increasemuscle mass, muscle strength, modulate the levels of muscle specificenzymes and/or stimulate myoblast proliferation for example. In apreferred embodiment, a GDF-8 inhibitor reduces clinical manifestationsof a GDF-8 associated disorder. A GDF-8 modulating agent can modulatepreadipocyte differentiation to adipocytes, decrease fat accumulation,decrease serum triglyceride levels, decrease serum cholesterol levels,modulate glucose metabolism, modulate bone density and reducehyperglycemia. A GDF-8 inhibitor may also be administered to anindividual in order to increase muscle mass, to increase or accelerategrowth, including muscle growth. A therapeutically effective amount of aGDF-8 inhibitor refers to an amount which is effective, upon single ormultiple dose administration to an individual at treating, preventing,curing, delaying, reducing the severity of, or ameliorating at least onesymptom of a disorder or recurring disorder, or prolonging the survivalof the subject beyond that expected in the absence of such treatment.

An individual with a GDF-8 associated disorder, an individual at riskfor developing a GDF-8 associated disorder, an individual undergoingtherapy with a GDF-8 modulating agent, and an individual who is acandidate for administration of a GDF-8 modulating agent, may be acandidate for the methods herein provided. The methods of the inventionmay detect or prevent a deleterious immune response, and/or assessefficacy, biological stability or suitability of use of a GDF-8modulating agent.

An individual having, or at risk for developing a GDF-8 associateddisorder such as a muscle-related disorder or a neuromuscular disorderis a candidate for the methods provided herein. Inhibition of a GDF-8activity increases muscle tissue individuals, including those sufferingfrom muscle-related disorders. A number of disorders are associated withfunctionally impaired muscle or nerve tissue, for example but notlimited to muscular dystrophies, amyotrophic lateral sclerosis (ALS),sarcopenia, cachexia, muscle wasting, muscle atrophy, or frailty.Muscular dystrophies include, for example, pseudohypertrophic,facioscapulohumeral, and limb-girdle dystrophies. Exemplary musculardystrophies include Duchennes' muscular dystrophy (Leyden-Mobius),Becker muscular dystrophy, Emery Dreifuss muscular dystrophy,limb-girdle muscular dystrophy, rigid spine syndrome, Ullrich syndrome,Fukuyama muscular dystrophy, Walker Warberg syndrome, muscle_eye_braindisease, facioscapulo-humeral muscular dystrophy (Landouzy-Dejerine),congenital muscular dystrophy, myotonic dystrophy (Steinart's Disease),and other myotonias and Gower's disease.

A GDF-8 associated muscle disorder also includes a disorder chosen frommuscle degeneration associated with cardiovascular disease, or secondaryto another disease or condition such as organ atrophy, organ failure,cancer, Acquired Immune Deficiency Syndrome (AIDS), bed rest,immobilization, prolonged lack of use, or other disease or condition arealso included in the term.

An individual having or at risk for developing adipose tissue disorders,e.g. obesity, cardiovascular disorders (when associated with muscle lossor muscle wasting) and disorders of insulin metabolism may be acandidate. Similarly, individuals having or at risk for developing, adisorder associated with a loss of bone, including osteoporosis,especially in the elderly and/or postmenopausal women,glucocorticoid-induced osteoporosis, osteopenia, osteoarthritis, andosteoporosis-related fractures are candidates for the treatment methodsprovided herein. Other GDF-8 associated conditions include metabolicbone disease and disorders characterized by low bone mass such as thosedue to chronic glucocorticoid therapy, premature gonadal failure,androgen suppression, vitamin D deficiency, secondaryhyperparathyroidism, nutritional deficiencies, and anorexia nervosa.

Examples of cardiovascular disorders include coronary artery disease(atherosclerosis), angina (including acute and unstable angina), heartattack, stroke (including ischemic stroke), hypertension associatedcardiovascular diseases, heart failure, congestive heart failure,coronary artery disease, hypertension, hyperlipidemia, peripheralarterial disease, and peripheral vascular disease. Examples of disordersof insulin metabolism include conditions associated with aberrantglucose homeostasis, type 2 diabetes, prediabetes, impaired glucosetolerance, dyslipidemia, metabolic syndrome (e.g. syndrome X), andinsulin resistance induced by trauma such as burns or nitrogenimbalance.

The term “GDF-8” refers to a specific growth and differentiationfactor-8, and may also be called “myostatin.” The term refers to thefull-length unprocessed precursor form of GDF-8 as well as the matureand propeptide forms resulting from post-translational cleavage. Unlessotherwise specified as “inactive,” a “GDF-8 protein” retains one or moreGDF-8 biological activities. The term also refers to any fragments andvariants of GDF-8 that maintain at least one biological activityassociated with mature GDF-8, as discussed herein, including sequencesthat have been modified. The amino acid sequence of mature human GDF-8is provided in SEQ ID NO: 1 The present invention relates to GDF-8 fromall vertebrate species, including, but not limited to, human, bovine,chicken, mouse, rat, porcine, ovine, turkey, baboon, and fish (forsequence information, see, e.g., McPherron et al., Proc. Nat. Acad. Sci.U.S.A. 94:12457-12461 (1997)).

The term “GDF-8 activity” refers to one or more physiologicallygrowth-regulatory or morphogenetic activities associated with activeGDF-8 protein. For example, active GDF-8 is a negative regulator ofskeletal muscle mass. Active GDF-8 can also modulate the production ofmuscle-specific enzymes (e.g., creatine kinase), stimulate myoblastproliferation, and modulate preadipocyte differentiation to adipocytes.“GDF-8 activity” includes “GDF-8 binding activity.” For example, matureGDF-8 specifically binds to the propeptide region of GDF-8, to ActRIIB,to a GDF-8 receptor, to activin, to follistatin, tofollistatin-domain-containing proteins, to GASP-1, and to otherproteins. A GDF-8 inhibitor, such as an antibody or antigen bindingprotein or portion thereof, may reduce one or more of these bindingactivities. The biological activities of GDF-8 are well known to thoseof skill in the art, see, for example, U.S. Patent Application No.2004/0223966 at examples 5-6 and 8-12.

A “GDF-8 associated disorder” is a disorder or condition in which asubject would benefit from the administration of a GDF-8 modulator, suchas a GDF-8 inhibitor. A GDF-8 associated disorder includes a medicaldisorder such as a muscle-related or neuromuscular disorder orcondition, for example, muscular dystrophy, amyotrophic lateralsclerosis (ALS), sarcopenia, cachexia, muscle wasting, muscle atrophy,or muscle degeneration, including wasting, atrophy, or frailty. Musculardystrophies include, for example, pseudohypertrophic,facioscapulohumera, and limb-girdle muscular dystrophies. Exemplarymuscular dystrophies include Duchenne's muscular dystrophy(Leyden-Möbius), Becker muscular dystrophy, Emery Dreifuss musculardystrophy, limb girdle muscular dystrophy, rigid spine syndrome, Ullrichsyndrome, Fukuyama muscular dystrophy, Walker Warburg syndrome, muscleeye brain disease, facioscapulohumeral muscular dystrophy(Landouzy-Dejerine), congenital muscular dystrophy, myotonic dystrophy(Steinert's disease), and othermyotonias, and Gowers disease.

Muscle degeneration associated with cardiovascular disease, or secondaryto another disease or condition such as organ atrophy, organ failure,cancer, Acquired Immune Deficiency Syndrome (AIDS), bed rest,immobilization, prolonged lack of use, or other disease or condition isalso included in the term.

GDF-8 associated disorders also include adipose tissue disorders (e.g.,obesity), cardiovascular diseases or disorders (when associated withmuscle loss or muscle wasting), and disorders of insulin metabolism.GDF-8 associated disorders also include disorders associated with a lossof bone, including osteoporosis, especially in the elderly and/orpostmenopausal women, glucocorticoid-induced osteoporosis, osteopenia,osteoarthritis, and osteoporosis-related fractures. Other conditionsinclude metabolic bone diseases and disorders characterized by low bonemass, such as those due to chronic glucocorticoid therapy, prematuregonadal failure, androgen suppression, vitamin D deficiency, secondaryhyperparathyroidism, nutritional deficiencies, and anorexia nervosa.

Examples of cardiovascular disorders include coronary artery disease(atherosclerosis), angina (including acute angina and unstable angina),heart attack, stroke (including ischemic stroke), hypertensionassociated cardiovascular diseases, heart failure, congestive heartfailure, coronary artery disease, hypertension, hyperlipidemia,peripheral arterial disease, and peripheral vascular disease. Examplesof disorders of insulin metabolism include conditions associated withaberrant glucose homeostasis, type 2 diabetes, prediabetes, impairedglucose tolerance, dyslipidemia, metabolic syndrome (e.g., syndome X),and insulin resistance induced by trauma such as burns or nitrogenimbalance.

The terms “GDF-8 latent complex” refers to the complex of proteinsformed between the mature GDF-8 homodimer and the GDF-8 propeptide. Itis believed that the two GDF-8 propeptides associate with the twomolecules of mature GDF-8 in the homodimer to form an inactivetetrameric complex. The latent complex may include other GDF inhibitorsin place of or in addition to one or more of the GDF-8 propeptides.

The term “mature GDF-8” refers to the protein that is cleaved from thecarboxy-terminal domain of the GDF-8 precursor protein. The mature GDF-8may be present as a monomer, homodimer, or in a GDF-8 latent complex.Depending on conditions, mature GDF-8 may establish equilibrium betweenany or all of these different forms. In its biologically active form,the mature GDF-8 is also referred to as “active GDF-8.” Biologicallyactive GDF-8 is not in a GDF-8 latent complex. The term also refers toany fragments and variants of GDF-8 that maintain at least onebiological activity associated with mature GDF-8, as discussed herein,including sequences that have been modified.

The term “GDF-8 propeptide” refers to the polypeptide that is cleavedfrom the amino-terminal domain of the GDF-8 precursor protein. The GDF-8propeptide is capable of binding to the propeptide binding domain on themature GDF-8. The GDF-8 propeptide forms a complex with the mature GDF-8homodimer. It is believed that two GDF-8 propeptides associate with twomolecules of mature GDF-8 in the homodimer to form an inactivetetrameric complex, called a “latent complex.” The latent complex mayinclude other GDF inhibitors in place of or in addition to one or moreof the GDF-8 propeptides.

The term “GDF-8 modulating agent” includes any agent capable ofmodulating activity, expression, processing, or secretion of GDF-8, or apharmaceutically acceptable derivative thereof. GDF-8 modulating agentswill increase or decrease one or more GDF-8 activities. A GDF-8modulator, including a “GDF-8 inhibitor,” may be used to treat adipocytedisorders, glucose metabolism-related disorders, or bone disorders, forexample. Biological derivatives of a GDF-8 modulating agent areencompassed by the term. In certain embodiments, a GDF-8 modulatingagent or inhibitor will affect binding of GDF-8 to one or more of itsphysiological binding partners, including, but not limited to a receptor(e.g. ActRIIB), a follistatin-domain containing protein (e.g.follistatin, FLRG, GASP-1, GASP-2), or a GDF-8 protein such as the GDF-8propeptide and mutants and derivatives thereof. GDF-8 modulating agentsinclude, for example, antibodies that specifically bind to GDF-8(including MYO-029, MYO-028, MYO-022, JA-16, and fragments andderivatives thereof), antibodies that specifically bind to a GDF-8receptor (see, e.g., U.S. Pat. No. 6,656,475, U.S. Patent Pub. No.2004/0077053-A1), modified soluble receptors (including receptor fusionproteins, such as an ActRIIB-Fc fusion protein), other proteins thatspecifically bind to GDF-8 or BMP-11 (such as the GDF-8 or BMP-11propeptide, mutants and derivatives of the GDF-8 propeptide,follistatin, follistatin-domain containing proteins, and Fc fusions ofthese proteins), proteins binding to the GDF-8 receptor and Fc fusionsof these proteins, and mimetics are included. Non-proteinaceousinhibitors (such as nucleic acids) are also encompassed by the termGDF-8 inhibitor. GDF-8 inhibitors include proteins, antibodies,peptides, peptidomimetics, ribozymes, anti-sense oligonucleotides,double-stranded RNA (including siRNA or microRNA) and other smallmolecules, which specifically inhibit GDF-8.

The term “individual” refers to any vertebrate animal, including amammal, bird, reptile, amphibian, or fish. The term “mammal” includesany animal classified as such, male or female, including humans,non-human primates, monkeys, dogs, horses, cats, sheep, pigs, goats,cattle, etc. Examples of non-mammalian animals include chicken, turkey,duck, goose, fish (such as salmon, catfish, bass, zebrafish, and trout),and frogs. An individual may be chosen from humans, or domesticated,feedstock, livestock, zoo, sports, racing, or pet animals, for example.

The terms “inhibit” and “inhibitory” refer to a reduction is one or moreactivities of GDF-8 by a GDF-8 inhibitor, relative to the activity ofGDF-8 in the absence of the same inhibitor. The reduction in activity ispreferably at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% orhigher. In certain embodiments, the activity of GDF-8 when affected byone or more of the presently disclosed inhibitors, is reduced at least50%, preferable at least about 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%,76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 99%, andeven more preferable at least 95% to 100%. The terms “neutralize” and“neutralizing” refer to a reduction one or more GDF-8 activities by atleast 80%, 85%, 90% or 95%. Inhibition of GDF-8 activity can be measuredfor example, in the pGL3(CAGA)12 reporter gene assays as described inThies et al. Growth Factors 18:251-259 (2001) or in ActRIIB receptorassays as illustrated in the Examples below.

The term “isolated” refers to a molecule that is substantially separatedfrom its natural environment. For instance, an isolated protein is onethat is substantially separated from the cell or tissue source fromwhich it is derived.

A label may also be an enzyme, for example an enzyme that converts asubstrate, such as a peroxidase (e.g., horseradish peroxidase), alkalinephosphatase, glucose oxidase, and β-galactosidase. Peroxidase, whenincubated with soluble substrates (e.g., 3,3′,5,5′ tetramethylbenzidine(TMB), o-phenylenediamine (OPD),2,2′-azino-di[3-ethyl-benzthiazoline]sulfonate (ABTS), luminol,polyphenols, acridine esters, and luciferin), results in a chromogenicor luminescent change in the substrate that can be detectedspectroscopically. Typically, after a fixed incubation period with thesubstrate, the reaction is quenched (e.g., by acidification), and theresult is quantified by measuring optical density (absorbance) orluminescence. Absorbance results can be compared with the OD values inthe linear range for chomogenic reactions, and luminescent immunoassaysare measured in relative light units (RLU).

A label may also be biotin, a hapten, or an epitope tag (e.g.,histidine, HA (hemagglutinin peptide), maltose binding protein, AviTag®,or glutathione-S-transferase), which can be detected by the addition ofa labeled detection agent that interacts with the label associated withthe GDF 8 modulating agent or detection agent. A biotin-labeled(“biotinylated”) detection agent may be detected through its interactionwith an avidin-enzyme conjugate, e.g., avidin-horseradish peroxidase,after sequential incubation with the avidin-enzyme conjugate and asuitable chromogenic or luminescent substrate. Europium is also a label.

The term “peptide mimetic”, as used herein, refers to a peptide thatbiologically mimics active determinants on hormones, cytokines, enzymesubstrates, viruses or other bio-molecules, and may antagonize,stimulate, or otherwise modulate the physiological activity of a naturalligand. Peptide mimetic are preferably defined as compounds which have asecondary structure like a peptide and optionally further structuralcharacteristics; their mode of action is largely similar or identical tothe mode of action of the native peptide however their activity (e.g. asan antagonist or an inhibitor) can be modified as compared with thenative peptide especially receptors or enzymes. Moreover, they canimitate the effect of the native peptide (agonist). Throughout thisspecification the term “peptide mimetic” refers to a molecule whichbecause of its structural properties is capable of mimicking thebiological functions of either functional GDF8 or non-functional GDF8.In the present invention, a fragment of GDF8 that comprises abinding/dimerization domain of GDF8 is proposed to function as adominant negative to GDF8. A peptide mimetic, per se, of abinding/dimerization domain of GDF8 could be present in multimolarexcess and can “outcompete” wild type GDF8 and form heterodimers withthe wild type molecules thereby acting as a dominant negative of thebiological function of GDF8. In this sense, GDF8 function would bedisrupted thus relieving the inhibition of muscle growth. One example ofan in vivo biological assay of such growth promoting GDF8 mimeticactivity is the enhancement of skeletal muscle mass in normal mice orother test animal by administration of an effective amount of at lestone growth promoting mimetic of the present invention.

The term “purified” refers to a molecule that is substantially free ofother material that associates with the molecule in its naturalenvironment. For instance, a purified protein is substantially free ofthe cellular material or other proteins from the cell or tissue fromwhich it is derived. The term refers to preparations where the isolatedprotein is sufficiently pure to be administered as a therapeuticcomposition, or at least 70% to 80% (w/w) pure, more preferably, atleast 80%-90% (w/w) pure, even more preferably, 90-95% pure; and, mostpreferably, at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.

The term “siRNA”, as used herein, refers to small interfering RNAmolecules that can be used to silence the expression of target genes.The siRNA can be dsRNA having 19-25 nucleotides. siRNAs can be producedendogenously by degradation of longer dsRNA molecules by an RNaseIII-related nuclease called Dicer. siRNAs can also be introduced into acell exogenously, or by transcription of an expression construct. Onceformed, the siRNAs assemble with protein components intoendoribonucleases-containing complexes known as RNA-induced silencingcomplexes (RISCs). An ATP-generated unwinding of the siRNA activates theRISCs, which in turn target the complementary mRNA transcript byWatson-Crick base-pairing, thereby cleaving and destroying the mRNA.Cleavage of the mRNA takes place near the middle of the region bound bythe siRNA strand. This sequence specific mRNA degradation results ingene silencing.

At least two ways can be employed to achieve siRNA-mediated genesilencing. First, siRNAs can be synthesized in vitro and introduced intocells to transiently suppress gene expression. siRNAs are duplexes ofshort mixed oligonucleotides which can include, for example, 19 RNAsnucleotides with symmetric dinucleotide 3′ overhangs. Using synthetic 21bp siRNA duplexes (e.g., 19 RNA bases followed by a UU or dTdT 3′overhang), sequence specific gene silencing can be achieved in mammaliancells. These siRNAs can specifically suppress targeted gene translationin mammalian cells without activation of DNA-dependent protein kinase(PKR) by longer double-stranded RNAs (dsRNA), which may result innon-specific repression of translation of many proteins.

Second, siRNAs can be expressed in vivo from vectors. This approach canbe used to stably express siRNAs in cells or transgenic animals. siRNAexpression vectors are engineered to drive siRNA transcription frompolymerase III (pol III) transcription units. Pol III transcriptionunits are suitable for hairpin siRNA expression because they deploy ashort AT rich transcription termination site that leads to the additionof 2 bp overhangs (e.g., UU) to hairpin siRNAs—a feature that is helpfulfor siRNA function. The Pol III expression vectors can also be used tocreate transgenic mice that express siRNA.

siRNAs can be also be expressed in a tissue-specific manner. Under thisapproach, long dsRNAs are first expressed from a promoter (such as CMV(pol II)) in the nuclei of selected cell lines or transgenic mice. Thelong dsRNAs are processed into siRNAs in the nuclei (e.g., by Dicer).The siRNAs exit from the nuclei and mediate gene-specific silencing. Asimilar approach can be used in conjunction with tissue-specific (polII) promoters to create tissue-specific knockdown mice.

The term “small molecule” refers to compounds that are notmacromolecules. See, e.g., Karp, (2000) Bioinformatics Ontology16:269-85; Verkman, (2004) AJP-Cell Physiol. 286:465-74. Thus, smallmolecules are often considered those compounds that are less than onethousand daltons (e.g., Voet and Voet, Biochemistry, 2nd ed., ed. N.Rose, Wiley and Sons, New York, 14 (1995). For example, Davis et al.((2005) Proc. Natl. Acad. Sci. USA 102:5981-86), use the phrase smallmolecule to indicate folates, methotrexate, and neuropeptides, whileHalpin and Harbury ((2004) PLos Biology 2:1022-30), use the phrase toindicate small molecule gene products, i.e., DNAs, RNAs and peptides.Examples of natural small molecules include cholesterols,neurotransmitters, and siRNAs; synthesized small molecules includevarious chemicals listed in numerous commercially available smallmolecule databases, e.g., FCD (Fine Chemicals Database), SMID (SmallMolecule Interaction Database), ChEBI (Chemical Entities of BiologicalInterest), and CSD (Cambridge Structural Database) (see, e.g., Alfaranoet al. (2005) Nuc. Acids Res. Database Issue 33:D416-24).

The terms “specific binding,” “specifically binds,” and the like, meanthat two or more molecules form a complex that is measurable underphysiologic or assay conditions and is selective. An antibody or antigenbinding protein or other inhibitor is said to “specifically bind” to aprotein if, under appropriately selected conditions, such binding is notsubstantially inhibited, while at the same time non-specific binding isinhibited. Specific binding is characterized by a high affinity and isselective for the compound or protein. Nonspecific binding usually has alow affinity. Binding in IgG antibodies for example is generallycharacterized by an affinity of at least about 10-7 M or higher, such asat least about 10-8 M or higher, or at least about 10-9 M or higher, orat least about 10-10 or higher, or at least about 10-11 M or higher, orat least about 10-12 M or higher. The term is also applicable where,e.g., an antigen-binding domain is specific for a particular epitopethat is not carried by numerous antigens, in which case the antibody orantigen binding protein carrying the antigen-binding domain willgenerally not bind other antigens.

Certain methods require high affinity for specific binding, whereasother methods, such as a surface plasmon resonance assay, may detectless stable complexes and lower affinity interactions. If necessary,non-specific binding can be reduced without substantially affectingspecific binding by varying the binding conditions. Such conditions areknown in the art, and a skilled artisan using routine techniques canselect appropriate conditions. The conditions are usually defined interms of concentration of the binding partners, ionic strength of thesolution, temperature, time allowed for binding, concentration ofnon-related molecules (e.g., serum albumin, milk casein), etc. Exemplarybinding conditions are set forth in the Examples below.

The term “specific GDF8 antagonist” or “specific GDF8 inhibitor”includes any agent capable of inhibiting, reducing and/or neutralizingactivity, expression, processing, or secretion of GDF8 but does notsignificantly inhibit, reduce and/or neutralize the activity,expression, processing, or secretion of other proteins, e.g., of theTGF-β superfamily, e.g., BMP11. Such inhibitors include macromoleculesand small molecules, e.g., proteins, antibodies, peptides,peptidomimetics, siRNA, ribozymes, anti-sense oligonucleotides,double-stranded RNA, and other small molecules, that specificallyinhibit GDF8 activity. Such inhibitors are said to specifically“antagonize”, (e.g., “inhibit,” “decrease,” “reduce” or “neutralize”)the biological activity of GDF8. A GDF-8 inhibitor will inhibit orneutralize or reduce at least one biological acitivy of GDF-8, such as aphysiological, growth-regulatory, or morphogenic activity associatedwith active GDF-8 protein. For example, GDF-8 is a negative regulator ofskeletal muscle growth. A GDF-8 inhibitor can increase muscle mass,increase muscle strength, modulate the levels of muscle specificenzymes, stimulate myoblast proliferation, modulate preadipocytedifferentiation to adipocytes, decrease fat accumulation, decreaseserumtriglyceride levels, decrease serum cholesterol levels, modulateglucose metabolism and/or reduce hyperglycemia.

The term “treatment” is used interchangeably herein with the term“therapeutic method” and refers to both therapeutic treatment andprophylactic/preventative measures. The term treatment is also definedas being able to ameliorate, treat or prevent a disorder. Those in needof treatment may include individuals already having a particular medicaldisorder as well as those who may ultimately acquire the disorder (i.e.,those needing preventive measures).

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Sambrook, et al., Molecular Cloning; A Laboratory Manual, Second Edition(1989); DNA Cloning, Volumes I And II (D. N Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gait ed., 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture(R. I. Freshney ed. 1986); Immobilized Cells And Enzymes (IRL Press,1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); theseries, Methods In Enzymology (Academic Press, Inc.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol.155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker,eds. (1987), Immunochemical Methods In Cell And Molecular Biology(Academic Press, London), Scopes, (1987), Protein Purification:Principles And Practice, Second Edition (Springer-Verlag, N.Y.), andHandbook Of Experimental Immunology, Volumes I IV (D. M. Weir and C. C.Blackwell eds. (1986).

Epitopes Specific to GDF8 and Antagonists Thereto

Epitope mapping using specific GDF8 antibodies and overlapping 13 aminoacid peptides of human GDF8 revealed candidate epitopes specific to GDF8that may be targeted for the specific antagonism of GDF8 (Example 4.2).Based on this approach, five independent epitope(s) specific to GDF8were identified. The present invention provides these epitopes(including peptide mimetics thereof), polynucleotides encoding theepitopes, inhibitory polynucleotides thereto, and antibodies relatedthereto as specific antagonists to GDF8 activity.

Epitopes Specific to GDF8 and Peptide Mimetics Thereof

The present invention provides novel isolated and purified polypeptideshomologous to epitopes, which may be biologically characterized as beingspecific to GDF8, and thus, are referred to herein as epitope(s)specific to GDF8. It is part of the invention that peptide mimetics ofthese GDF8 specific epitopes may be used as GDF8 antagonists, i.e., toantagonize GDF8 activity, e.g., GDF8 binding to its receptor.

For example, the invention provides purified and isolatedpolynucleotides encoding five binding domains specific to GDF8 (whichmay include ALK4/ALK4 receptor binding sites of GDF8), and which mayalso function as GDF8 receptor antagonists/peptide mimetics, hereindesignated “GE1,” “GE2,” “GE3,” “GE4,” and “GE5.” Nucleic acid accordingto the present invention may comprise DNA or RNA and may be wholly orpartially synthetic. Reference to a nucleotide sequence as set outherein encompass DNA molecules with the specified sequences or genomicequivalents (e.g., complementary sequences), as well as RNA moleculeswith the specified sequences where T is substituted with U, unlesscontext requires otherwise. Preferred DNA sequences of the inventioninclude genomic and cDNA sequences and chemically synthesized DNAsequences.

Nucleotide sequences of cDNAs encoding human GE1, GE2, GE3, GE4, andGE5, designated human cDNA, are set forth as SEQ ID NOs:3, 5, 7, 9, and11, respectively. Polynucleotides of the present invention also includepolynucleotides that hybridize under stringent conditions topolynucleotides having and/or consisting essentially of the nucleotidesequences set forth as SEQ ID NOs: 3, 5, 7, 9, and 11, or complementsthereof, and/or encode polypeptides that retain substantial biologicalactivity of GE1, GE2, GE3, GE4, or GE5, respectively. Polynucleotides ofthe present invention also include continuous portions of the nucleotidesequences set forth as SEQ ID NOs: 3, 5, 7, 9, and 11 comprising atleast 12 consecutive nucleotides.

The amino acid sequences of human GE1, GE2, GE3, GE4, GE5, and mimeticpolypeptides thereto are set forth as SEQ ID NOs: 4, 6, 8, 10 and 12,respectively. Polypeptides of the present invention also includepolypeptides with an amino acid sequence having and/or consistingessentially of continuous portions of any of the sequences set forth asSEQ ID NOs: 4, 6, 8, 10 and 12, comprising at least 4 consecutive aminoacids. Polypeptides of the invention also include any of the sequencesset forth as SEQ ID NOs: 4, 6, 8, 10 and 12, including continuousportions thereof, wherein one or more of the L-amino acids are replacedwith their corresponding D-amino acids. Polypeptides of the presentinvention also include active fragments of SEQ ID NOs: 4, 6, 8, 10 and12, i.e., any continuous portion of any of the sequences set forth asSEQ ID NOs: 4, 6, 8, 10 and 12 that retains substantial biologicalactivity of full-length human GE1, GE2, GE3, GE4, or GE5, i.e., anyfragment of SEQ ID NOs: 4, 6, 8, 10 and 12 that is an binding domainspecific for GDF8 and/or to which a mimetic peptide thereto may be aspecific antagonist to GDF8 activity. Additionally, a polypeptide of theinvention may be acetylated and/or amide blocked using well-knownmethods. Polynucleotides of the present invention also include, inaddition to those polynucleotides described above, polynucleotides thatencode any of the amino acid sequences set forth as SEQ ID NOs: 4, 6, 8,10 and 12 and continuous portions thereof, and that differ from thepolynucleotides of human origin described above only due to thewell-known degeneracy of the genetic code.

The invention also provides purified and isolated polynucleotidesencoding cyclized mimetic peptides to the epitope(s) specific to GDF8,e.g., GE1, GE2, GE3, GE4, and GE5. Preferred DNA sequences of theinvention include genomic and cDNA sequences and chemically synthesizedDNA sequences. One of skill in the art will recognize that the presentinvention also includes other cyclized molecules, such as cyclizedmimetic peptides based on other binding domains specific to GDF8.Additionally, a cyclized mimetic peptide of the invention may beacetylated and/or amide blocked using well-known methods.

Antibodies Specific to GDF8

The present disclosure provides novel antibodies (e.g., antibody orantigen binding protein fragments) that specifically interact with GDF8.Nonlimiting illustrative embodiments of such antibodies are termed RK22.The antibodies of the invention possess unique and beneficialcharacteristics. First, these antibodies are capable of binding matureGDF8 with high affinity (Example 2). Second, the disclosed antibodiesspecifically interact with GDF8, i.e., the antibodies of the inventiondo not bind with high affinity to other members of the TGF-β subfamily,e.g., BMP11 (Example 2). Third, the antibodies of the invention inhibitGDF8 activity in vitro and in vivo as demonstrated (Example 3). Fourth,the disclosed antibodies may inhibit GDF8 activity associated withnegative regulation of skeletal muscle mass and bone density (Example3).

In one embodiment, the presently disclosed antibodies are capable ofspecifically interacting with GDF8; i.e., it is contemplated that theantibodies will not extensively react with other proteins, for example,those belonging to the TGF-β superfamily such as BMP11, activin,mullerian-inhibiting substance, glial-derived neurotrophic factor, orgrowth and differentiation factors other than GDF8. In one non-limitingembodiment of the invention, a specific GDF8 antibody or antigen bindingprotein of the invention binds GDF8 with 5-10 fold greater preferencethan it binds BMP11. In a nonlimiting embodiment of the invention, aspecific anti-GDF8 antibody or antigen binding protein of the inventionbinds GDF8 with 10-100 fold greater preference than it binds BMP11. Inone nonlimiting embodiment of the invention, a specific anti-GDF8antibody or antigen binding protein of interest binds GDF8 with 100-1000fold greater preference than it binds BMP11. In another embodiment, aspecific anti-GDF8 antibody or antigen binding protein of the inventionbinds to epitope(s) specific to GDF8, including those disclosed herein(e.g., epitope(s) specific to GDF8 having and/or consisting essentiallyof an amino acid sequence set forth as SEQ ID NOs.: 4, 6, 8, and 12, oractive fragments thereof). In one embodiment of the invention, thecontemplated antibodies specifically interact with the predictedALK4/ALK5 binding site of mature GDF8, e.g., GDF8 epitopes with an aminoacid sequence set forth as SEQ ID NOs:4, 6 or 8.

One of ordinary skill in the art will recognize that the antibodies ofthe invention may be used to detect, measure, and inhibit GDF8 proteinsderived from various species, e.g., those described in the presentspecification. The percent identity is determined by standard alignmentalgorithms such as, for example, Basic Local Alignment Tool (BLAST)described in Altschul et al. ((1990) J. Mol. Biol. 215:403-10), thealgorithm of Needleman et al. ((1970) J. Mol. Biol. 48:444-53), or thealgorithm of Meyers et al. ((1988) Comput. Appl. Biosci. 4:11-17). Ingeneral, the antibodies and antibody or antigen binding proteinfragments of the invention can be used with any protein that retainssubstantial GDF8 biological activity and comprises an amino acidsequence that is at least about 70%, 80%, 90%, 95%, or more identical toany sequence of at least 100, 80, 60, 40, 20, or 15 contiguous aminoacids of the mature form of GDF8 set forth as SEQ ID NO:1.

Intact antibodies, also known as immunoglobulins, are typicallytetrameric glycosylated proteins composed of two light (L) chains ofapproximately 25 kDa each, and two heavy (H) chains of approximately 50kDa each. Two types of light chain, termed lambda and kappa, exist inantibodies. Depending on the amino acid sequence of the constant domainof heavy chains, immunoglobulins are assigned to five major classes: A,D, E, G, and M, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.Each light chain is composed of an N-terminal variable (V) domain (VL)and a constant (C) domain (CL). Each heavy chain is composed of anN-terminal V domain (VH), three or four C domains (CHs), and a hingeregion. The CH domain most proximal to VH is designated CH1. The VH andVL domains consist of four regions of relatively conserved sequencesnamed framework regions (FR1, FR2, FR3, and FR4), which form a scaffoldfor three regions of hypervariable sequences (complementaritydetermining regions, CDRs). The CDRs contain most of the residuesresponsible for specific interactions of the antibody or antigen bindingprotein with the antigen. CDRs are referred to as CDR1, CDR2, and CDR3.Accordingly, CDR constituents on the heavy chain are referred to as H1,H2, and H3, while CDR constituents on the light chain are referred to asL1, L2, and L3. CDR3 is the greatest source of molecular diversitywithin the antibody or antigen binding protein-binding site. H3, forexample, can be as short as two amino acid residues or greater than 26amino acids. The subunit structures and three-dimensional configurationsof different classes of immunoglobulins are well known in the art. For areview of the antibody structure, see Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Eds. Harlow et al., 1988. One of skill inthe art will recognize that each subunit structure, e.g., a CH, VH, CL,VL, CDR, and/or FR structure, comprises active fragments. For example,active fragments may consist of the portion of the VH, VL, or CDRsubunit that binds the antigen, i.e., the antigen-binding fragment, orthe portion of the CH subunit that binds to and/or activates an Fcreceptor and/or complement.

Nonlimiting examples of binding fragments encompassed within the term“antibody or antigen binding protein fragment” used herein include: (i)an Fab fragment, a monovalent fragment consisting of the VL, VH, CL andCH1 domains; (ii) an F(ab′)2 fragment, a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region;(iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fvfragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment, which consists of a VH domain; and (vi) anisolated CDR. Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they may be recombinantlyjoined by a synthetic linker, creating a single protein chain in whichthe VL and VH domains pair to form monovalent molecules (known as singlechain Fv (scFv)). The most commonly used linker is a 15-residue(Gly4Ser)₃ peptide, but other linkers are also known in the art. Singlechain antibodies are also intended to be encompassed within the terms“antibody or antigen binding protein,” or “antigen-binding fragment” ofan antibody.

These antibodies are obtained using conventional techniques known tothose skilled in the art, and the fragments are screened for utility inthe same manner as intact antibodies. Antibody diversity is created bymultiple germline genes encoding variable domains and a variety ofsomatic events. The somatic events include recombination of variablegene segments with diversity (D) and joining (J) gene segments to make acomplete VH domain, and the recombination of variable and joining genesegments to make a complete VL domain. The recombination process itselfis imprecise, resulting in the loss or addition of amino acids at theV(D)J junctions. These mechanisms of diversity occur in the developing Bcell prior to antigen exposure. After antigenic stimulation, theexpressed antibody genes in B cells undergo somatic mutation. Based onthe estimated number of germline gene segments, the random recombinationof these segments, and random VH-VL pairing, up to 1.6×107 differentantibodies may be produced (Fundamental Immunology, 3rd ed. (1993), ed.Paul, Raven Press, New York, N.Y.). When other processes that contributeto antibody diversity (such as somatic mutation) are taken into account,it is thought that upwards of 1×1010 different antibodies may begenerated (Immunoglobulin Genes, 2nd ed. (1995), eds. Jonio et al.,Academic Press, San Diego, Calif.). Because of the many processesinvolved in generating antibody diversity, it is unlikely thatindependently derived monoclonal antibodies with the same antigenspecificity will have identical amino acid sequences.

Thus, the present invention provides novel antibodies that specificallyinteract with GDF8 i.e., specific GDF8 antibodies. The antibody orantigen binding protein fragments of the invention, e.g., structurescontaining a CDR, will generally be an antibody or antigen bindingprotein heavy or light chain sequence, or an active fragment thereof, inwhich the CDR is placed at a location corresponding to the CDR ofnaturally occurring VH and VL. The structures and locations ofimmunoglobulin variable domains, e.g., CDRs, may be defined usingwell-known numbering schemes, e.g., the Kabat numbering scheme, theChothia numbering scheme, a combination of Kabat and Chothia (AbM), etc.(see, e.g., Sequences of Proteins of Immunological Interest, U.S.Department of Health and Human Services (1991), eds. Kabat et al.;Al-Lazikani et al. (1997) J. Mol. Bio. 273:927-948).

Thus, the present invention further provides novel CDRs. The structurefor carrying a CDR of the invention will generally be a polypeptide,e.g., an antibody or antigen binding protein heavy or light chainsequence or a substantial portion thereof, in which the CDR is locatedat a position corresponding to the CDR of naturally occurring VH and VLdomains. The structures and locations of immunoglobulin variable domainsmay be determined as described in, e.g., Kabat et al., supra andAl-Lazikani et al., supra.

Antibody or antigen binding protein molecules capable of specificallyinteracting with the polypeptides of the present invention may beproduced by methods well known to those skilled in the art. For example,monoclonal antibodies can be produced by generation of hybridomas inaccordance with known methods. Hybridomas formed in this manner are thenscreened using standard methods, such as enzyme-linked immunosorbentassay (ELISA) and Biacore analysis, to identify one or more hybridomasthat produce an antibody that specifically interacts with GDF8 (e.g.,binds GDF8) and/or antagonizes (e.g., inhibits, reduces, and/orneutralizes) at least one GDF8 activity, (e.g., GDF8 binding to itsreceptor or other downstream GDF8 signaling events)).

Recombinant GDF8, naturally occurring GDF8, and antigenic peptidefragments of GDF8 may be used as the immunogen. An antigenic peptidefragment comprises at least six contiguous amino acids and encompassesan epitope such that an antibody raised against it forms a specificimmune complex with GDF8. Preferably, the antigenic peptide comprises atleast four amino acids residues. Additionally, it is preferable that theantigenic peptide fragment of GDF8 comprises an epitope specific to GDF8(e.g., a peptide having and/or consisting essentially of an amino acidsequence set forth as SEQ ID NOs: 4,6,8,10, 12, or active fragmentstransfer).

In one embodiment of the invention, a full-length GDF8 polypeptide maybe used as the immunogen, or, alternatively, antigenic peptide fragmentsof the polypeptide may be used. For example, the immunogen may be a GDF8specific epitope (e.g., an epitope specific to GDF8, and/or an epitopeof which specific anti-GDF8 antibodies and/or mimetic peptides directedthereto are specific antagonists of GDF8 signaling (e.g., one or more ofthe amino acid sequences of SEQ ID NOs: 4,6,8,10, 12, and activefragments thereof)) and/or a related peptide or cyclized peptide. Anantigenic peptide of a polypeptide of the present invention comprises atleast four continuous amino acid residues and encompasses an epitopesuch that an antibody raised against the peptide forms a specific immunecomplex with the polypeptide. Preferably, the antigenic peptidecomprises at least four amino acid residues, more preferably at leastsix amino acid residues, and even more preferably at least nine aminoacid residues.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal antibody to a polypeptide of the present invention may beidentified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) with apolypeptide of the present invention to thereby isolate immunoglobulinlibrary members that bind to the polypeptide. Techniques andcommercially available kits for generating and screening phage displaylibraries are well known to those skilled in the art. Additionally,examples of methods and reagents particularly amenable for use ingenerating and screening antibody or antigen binding protein displaylibraries can be found in the literature.

Polyclonal sera and antibodies may be produced by immunizing a suitablesubject with GDF8, its variants, and/or portion thereof, e.g., with aspecific GDF8 epitope of the present invention. The antibody titer inthe immunized subject may be monitored over time by standard techniques,such as with ELISA, or by using immobilized GDF8 or other marker protein(e.g., FLAG). If desired, the antibody molecules directed against apolypeptide of the present invention may be isolated from the subject orculture media and further purified by well known techniques, such asprotein A chromatography, to obtain an IgG fraction.

Additionally, chimeric, humanized, and single-chain antibodies to thepolypeptides of the present invention, comprising both human andnonhuman portions, may be produced using standard recombinant DNAtechniques. Humanized antibodies may also be produced using transgenicmice that are incapable of expressing endogenous immunoglobulin heavyand light chain genes, but that can express human heavy and light chaingenes.

Antibody or antigen binding protein molecules (which includes fragments)of the present invention (e.g., antibody or antigen binding proteinmolecules that specifically interact with GDF8) include, but are notlimited to, monoclonal RK22 antibody, variants thereof (e.g., humanizedvariants) and fragments thereof. Antibody or antigen binding proteinmolecules of the invention that specifically interact with GDF8 may alsobe specific GDF8 antagonists, and thus, these antibody or antigenbinding protein molecules may be useful in preventing or treating GDF8associated disorders, e.g., bone, muscle, adipose and glucosemetabolism-related pathologies.

Thus, the invention also provides purified and isolated polynucleotidesencoding the regions of specific GDF8 antibodies that may antagonize atleast one GDF8 activity e.g., RK22 and variants thereof. Preferred DNAsequences of the invention include genomic, cDNA, and chemicallysynthesized DNA sequences. As discussed above, the polynucleotidesencoding regions of an antibody or antigen binding protein of theinvention may comprise DNA or RNA and may be wholly or partiallysynthetic. Reference to a nucleotide sequence as set out hereinencompass DNA molecules with the specified sequences or genomicequivalents (e.g., complementary sequences), as well as RNA moleculeswith the specified sequence where T is substituted with U, unlesscontext requires otherwise.

The nucleotide sequences of the invention include those that encode thelight chain variable domain of murine RK22, e.g., the nucleotidesequence set forth as SEQ ID NO:15. The nucleotide sequences of theinvention also include those that encode the heavy chain variable domainof murine RK22, e.g., the nucleotide sequence set forth as SEQ ID NO:13.Polynucleotides of the present invention also include polynucleotidesthat hybridize under stringent conditions to polynucleotides havingand/or consisting essentially of the nucleic acid sequence(s)substantially set forth as SEQ ID NOs: 13 and 15, and complementsthereof, and/or that encode polypeptides that retain substantialbiological activity (i.e., active fragments) of the variable domains ofRK22. Polynucleotides of the present invention also include continuousportions of the sequence set forth as SEQ ID NOs: 13 and 15, comprisingat least 15 consecutive nucleotides.

The amino acid sequence of the light chain variable domains of murineRK22 is set forth as SEQ ID NO: 16. The amino acid sequences of theheavy chain variable domains of murine RK22 is set forth as SEQ ID NO:14. The amino acid sequence of humanized variable heavy and light chaindomains are set out in SEQ ID NOs: 17 and 18, respectively. The aminoacid sequences of the CDRs contained within the heavy chains of murineRK22 are set forth as SEQ ID NOs:19-21 and 25-27. The amino acidsequences of the CDRs contained within the light chains of murine RK22are set forth as SEQ ID NOs: 22-24 and 28-30. Polypeptides of thepresent invention also include continuous portions of any of thesequences substantially set forth as SEQ ID NOs: 14, 16, 17, 18, and19-30 comprising at least 5 consecutive amino acids. A preferredpolypeptide of the present invention includes active fragments as SEQ IDNOs: 14, 16, 17, 18, and 19-30, i.e., any continuous portion of anysequence set forth as SEQ ID NOs: 14, 16, 17, 18, and 19-30 retainingsubstantial biological activity of an antibody or antigen bindingprotein of the invention. In addition to those polynucleotides describedabove, the present invention also includes polynucleotides that encodean amino acid sequence substantially set forth as SEQ ID NOs: 14, 16,17, 18, and 19-30, or a continuous portion thereof, and that differ fromthe antibody or antigen binding protein polynucleotides described aboveonly due to the well-known degeneracy of the genetic code.

As described above, the CDRs contain most of the residues responsiblefor specific interactions with an antigen, and are contained within theVH and VL domains, i.e., the heavy chain variable domain and the lightchain variable domain, respectively. Consequently, provided that anantibody comprises at least one CDR of an antibody or antigen bindingprotein of the invention, e.g., a CDR comprising an amino acid sequenceselected from the amino acid sequences set forth as SEQ ID NOs: 19-30,or active antibody or antigen binding protein fragments thereof, it isan antibody of the invention, i.e., one that specifically interacts withGDF8 (e.g., binds to GDF8) and/or specifically antagonizes GDF8activity. Therefore, an embodiment of the invention includes antibodiesthat contain one or more CDRs that comprise(s) an amino acid sequenceselected from an amino acid sequence set forth as SEQ ID NOs: 19-30, oran amino acid sequence of active fragments thereof. Consequently, one ofskill in the art will recognize that the antibodies of the inventionincludes an antibody or antigen binding protein in which the CDRs of theVL chain are one or more CDRs of those set forth as SEQ ID NOs:22-24 and28-30, and/or the CDRs of the VH chain are one or more CDRs of those setforth as SEQ ID NOs:1921 and 25-27.

An antigen-binding fragment may be an Fv fragment, which consists of VHand VL domains. Thus, an Fv fragment of RK22 may constitute an antibodyof the invention, provided that the fragment specifically interacts withGDF8. One of skill in the art will recognize that any antibody orantigen binding protein fragment containing the Fv fragment of RK22 mayalso be an antibody of the invention. Additionally, any Fv fragment,scFv fragment, Fab fragment, or F(ab′)2 fragment, which contains one ormore CDRs having an amino acid sequence selected from the groupconsisting of the amino acid sequences set forth as SEQ ID NOs:19-30 mayalso be an antibody or antigen binding protein of the invention.

Certain embodiments of the invention comprise the VH and/or VL domain ofthe Fv fragment of RK22. Fragments of antibodies of the presentinvention, e.g., Fab, F(ab′)2, Fd, and dAb fragments, may be produced bycleavage of the antibodies e.g., RK22 in accordance with methods wellknown in the art. For example, immunologically active Fab and F(ab′)2fragments may be generated by treating the antibodies with an enzyme,e.g., papain and pepsin respectively.

Further embodiments comprise one or more CDRs (e.g., one or more CDRsset forth as SEQ ID NOs: 19-21 and 25-27) of any of the VH domains of anantibody disclosed herein (e.g., the VH domains of RK22 (set forth asSEQ ID NOs:14 and 17) and VL domains of an antibody disclosed herein(e.g., the VL domains of RK22 (set forth as SEQ ID NOs:16 and 18. Oneembodiment comprises an H3 fragment of the VH domain of RK22 (set forthas SEQ ID NO:21.

For convenience, the approximate positions of each CDR within the VH andVL domains are listed in Table 2.

TABLE 2 Approximate CDR position according to Kabat (not ital) or AbM(ital) definitions within variable domains of RK22 mouse and humanizedantibodies RK22 RK22 CDR SEQ ID NO: 14 SEQ ID NO: 17 H1 50-54 or 45-5426-35 H2 69-85 or 69-77 50-66 H3 116-128 or 116-128  99-109 RK22 RK22SEQ ID NO: 16 SEQ ID NO: 18 L1 44-60 or 44-60 24-40 L2 76-82 or 76-8256-62 L3 115-123 or 115-123  95-101

Presently disclosed antibodies may further comprise antibody or antigenbinding protein constant domains or parts thereof. For example, a VLdomain of the invention may be attached at its C-terminal end to anantibody or antigen binding protein light chain constant domain, e.g., ahuman Cκ or Cλ chain, preferably a Cλ chain. Similarly, a specificantigen-binding fragment based on a VH domain may be attached at itsC-terminal end to all or part of an immunoglobulin heavy chain derivedfrom any antibody isotype, e.g., IgG, IgA, IgE, and IgM, and any of theisotype subclasses, particularly IgG1 and IgG4. In exemplaryembodiments, antibodies comprise C-terminal fragments of heavy and lightchains of human IgG1λ. It is understood that, due to the degeneracy ofthe genetic code, DNA sequences listed in the Brief Description of theSequences 1 are merely representative of nucleic acids that encode theamino acid sequences, peptides, and antibodies of interest, and are notto be construed as limiting.

Certain embodiments of the invention comprise the VH and/or VL domain ofthe Fv fragment of RK22. Further embodiments comprise one or morecomplementarity determining regions (CDRs) of any of these VH and VLdomains. One embodiment comprises an H3 fragment of the VH domain ofRK22. The VH and VL domains of the invention, in certain embodiments,are germlined, i.e., the framework regions (FRs) of these domains arechanged using conventional molecular biology techniques to match theconsensus amino acid sequences of human germline gene products. This isalso known as a humanized or germlined antibody. In other embodiments,the framework sequences remain diverged from the germline. Humanizedantibodies may be produced using transgenic mice that are incapable ofexpressing endogenous immunoglobulin heavy and light chain genes, butare capable of expressing human heavy and light chain genes.

A further aspect of the invention provides methods for obtaining anantibody antigen-binding domain specific for GDF8. The skilled artisanwill appreciate that the antibodies of the invention are not limited tothe specific sequences of VH and VL domains as stated in Table 2, butalso include variants of these sequences that retain antigen bindingability. Such variants may be derived from the provided sequences usingtechniques known in the art. Amino acid substitution, deletions, oradditions, can be made in either the FRs or in the CDRs. While changesin the framework regions are usually designed to improve stability andreduce immunogenicity of the antibody, changes in the CDRs are usuallydesigned to increase affinity of the antibody for its target. Suchaffinity-increasing changes are typically determined empirically byaltering the CDR and testing the antibody. Such alterations can be madeaccording to the methods described in, e.g., Antibody Engineering, 2nded., ed. Borrebaeck, Oxford University Press, 1995.

Thus, the antibodies or antigen binding protein (or fragments thereof)of the invention also include those that specifically interact withGDF8, and have mutations in the constant domains of the heavy and lightchains. It is sometimes desirable to mutate and inactivate certainfragments of the constant domain. For example, mutations in the heavyconstant domain are sometimes desirable to produce antibodies withreduced binding to the Fc receptor (FcR) and/or complement; suchmutations are well known in the art. One of skill in the art will alsorecognize that the determination of which active fragments of the CL andCH subunits are necessary will depend on the application to which anantibody of the invention is applied. For example, the active fragmentsof the CL and CH subunits that are involved with their covalent link toeach other will be important in the generation of an intact antibody.

The method for making a VH domain that is an amino acid sequence variantof a VH domain set out herein comprises a step of adding, deleting,substituting or inserting one or more amino acids in the amino acidsequence of the presently disclosed VH domain, optionally combining theVH domain thus provided with one or more VL domains, and testing the VHdomain or VH/VL combination or combinations for specific interactionwith GDF8, and (preferably) testing the ability of such antigen-bindingdomain to modulate one or more GDF8-associated activities. The VL domainmay have an amino acid sequence that is substantially as set out herein.An analogous method may be employed in which one or more sequencevariants of a VL domain disclosed herein are combined with one or moreVH domains.

A further aspect of the invention provides a method of preparing anantigen-binding fragment that specifically interacts with GDF8. Themethod comprises: providing a starting repertoire of nucleic acidsencoding a VH domain which either include a CDR, e.g., CDR3, to bereplaced or a VH domain that lacks a CDR, e.g., CDR3, encoding region;combining the repertoire with a donor nucleic acid encoding a donor CDRof the invention (e.g., a donor nucleic acid encoding a CDR comprisingan active fragment of SEQ ID NO:14, 16, 17, 18 such that the donornucleic acid is inserted into the CDR, e.g., CDR3, region in therepertoire so as to provide a product repertoire of nucleic acidsencoding a VH domain; expressing the nucleic acids of the productrepertoire; selecting an antigen-binding fragment specific for GDF8; andrecovering the specific antigen-binding fragment or nucleic acidencoding it. Again, an analogous method may be employed in which a VLCDR (e.g., L3) of the invention is combined with a repertoire of nucleicacids encoding a VL domain, which either include a CDR to be replaced orlack a CDR encoding region.

A coding sequence of a CDR of the invention (e.g., CDR3) may beintroduced into a repertoire of variable domains lacking a CDR (e.g.,CDR3), using recombinant DNA technology. For example, Marks et al.((1992) Bio/Technology 10:779-83) describes methods of producingrepertoires of antibody or antigen binding protein variable domains inwhich consensus primers directed at or adjacent to the 5′ end of thevariable domain area are used in conjunction with consensus primers tothe third framework region of human VH genes to provide a repertoire ofVH variable domains lacking a CDR3. The repertoire may be combined witha CDR3 of a particular antibody. Using analogous techniques, theCDR3-derived sequences of the present invention may be shuffled withrepertoires of VH or VL domains lacking a CDR3, and the shuffledcomplete VH or VL domains combined with a cognate VL or VH domain toprovide specific antigen-binding fragments of the invention. Therepertoire may then be displayed in a suitable host system, such as thephage display system of WO 92/01047, so that suitable antigen-bindingfragments can be selected.

Analogous shuffling or combinatorial techniques are also disclosed byStemmer ((1994) Nature 370:389-91), who describes a technique inrelation to a β-lactamase gene but observes that the approach may beused for the generation of antibodies. A further alternative is togenerate novel VH or VL domains carrying a CDR-derived sequence of theinvention using random mutagenesis of one or more selected VH and/or VLgenes to generate mutations within the entire variable domain. Such atechnique is described in Gram et al. ((1992) Proc. Natl. Acad. Sci.U.S.A. 89:3576-80) by using error-prone PCR. Another method that may beused to generate novel antibodies or fragments thereof is to directmutagenesis to CDRs of VH or VL genes. Such techniques are disclosed inBarbas et al. ((1994) Proc. Natl. Acad. Sci. U.S.A. 91:3809-13) andSchier et al. ((1996) J. Mol. Biol. 263:551-67).

Similarly, one, two, or all three CDRs may be grafted into a repertoireof VH or VL domains, which are then screened for a specific bindingpartner or binding fragments specific for GDF8. A substantial portion ofan immunoglobulin variable domain will comprise at least the CDRs and,optionally, their intervening framework regions from the antibodyfragments as set out herein. The portion will also include at leastabout 50% of either or both of FR1 and FR4, the 50% being the C-terminal50% of FR1 and the N-terminal 50% of FR4. Additional residues at theN-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domains. For example, construction of specific antibody orantigen binding protein fragments of the present invention made byrecombinant DNA techniques may result in the introduction of N- orC-terminal residues encoded by linkers introduced to facilitate cloningor other manipulation steps. Other manipulation steps include theintroduction of linkers to join variable domains of the invention tofurther protein sequences including immunoglobulin heavy chains, othervariable domains (for example, in the production of diabodies) orprotein labels as discussed in more details below.

Although the embodiments illustrated in the Examples comprise a“matching” pair of VH and VL domains, the invention also encompassesbinding fragments containing a single variable domain, e.g., a dAbfragment, derived from either VH or VL domain sequences, especially VHdomains. In the case of either of the single chain specific bindingdomains, these domains may be used to screen for complementary domainscapable of forming a two-domain specific antigen-binding domain capableof binding GDF8. This may be achieved by phage display screening methodsusing the so-called hierarchical dual combinatorial approach asdisclosed in, e.g., WO 92/01047. In this technique, an individual colonycontaining either an H or L chain clone is used to infect a completelibrary of clones encoding the other chain (L or H) and the resultingtwo-chain specific antigen-binding domain is selected in accordance withphage display techniques, such as those described in that reference.This technique is also disclosed in Marks et al., supra.

Antibodies can be conjugated by chemical methods with radionuclides,drugs, macromolecules, or other agents, and may be made as fusionproteins comprising one or more CDRs of the invention.

An antibody or antigen binding protein fusion protein contains a VH-VLpair in which one of these chains (usually VH) and another protein aresynthesized as a single polypeptide chain. These types of productsdiffer from antibodies in that they generally have an additionalfunctional element, (e.g., the active moiety of a small molecule or theprincipal molecular structural feature of the conjugated or fusedmacromolecule).

In addition to the changes to the amino acid sequence outlined above,the antibodies can be glycosylated, pegylated, or linked to albumin or anonproteinaceous polymer. For instance, the presently disclosedantibodies may be linked to one of a variety of non-proteinaceouspolymers, e.g., polyethylene glycol, polypropylene glycol, orpolyoxyalkylenes. The antibodies may be chemically modified, e.g., toincrease their circulating half-life by covalent conjugation to apolymer. Exemplary polymers, and methods to attach them to peptides areknown in the art.

In other embodiments, the antibody or antigen binding protein may bemodified to have an altered glycosylation pattern (i.e., relative to theoriginal or native glycosylation pattern). As used herein, “altered”means having one or more carbohydrate moieties deleted, and/or havingone or more glycosylation sites added to the original antibody. Additionof glycosylation sites to the presently disclosed antibodies isaccomplished by well-known methods of altering the amino acid sequenceto contain glycosylation site consensus sequences. Another means ofincreasing the number of carbohydrate moieties on the antibodies is bychemical or enzymatic coupling of glycosides to the amino acid residuesof the antibody. Removal of any carbohydrate moieties present on theantibodies may be accomplished chemically or enzymatically as known inthe art.

Antibodies of the invention may also be tagged with a detectable orfunctional label such as 131I or 99Tc, which may be attached toantibodies of the invention using conventional chemistry known in theart. Labels also include enzyme labels such as horseradish peroxidase oralkaline phosphatase. Labels further include chemical moieties such asbiotin, which may be detected via binding to a specific cognatedetectable moiety, e.g., labeled avidin.

Antibodies in which CDR sequences differ only insubstantially from CDRsequences of the antibodies disclosed herein are encompassed within thescope of this invention. Insubstantial differences include minor aminoacid changes, e.g., substitutions of one or two out of any five aminoacids in the sequence of a CDR. Typically, an amino acid is substitutedby a related amino acid having similar charge, hydrophobicity, orstereochemical characteristics. Such substitutions would be within theordinary skills of an artisan. The structure framework regions (FRs) canbe modified more substantially than CDRs without adversely affecting thebinding properties of an antibody. Changes to FRs include, but are notlimited to, humanizing a nonhuman derived framework or engineeringcertain framework residues that are important for antigen contact or forstabilizing the binding site, e.g., changing the class or subclass ofthe constant domain, changing specific amino acid residues which mightalter an effector function such as Fc receptor binding (e.g., Lund etal. (1991) J. Immunol. 147:2657-62; Morgan et al. (1995) Immunology86:319-24), or changing the species from which the constant domain isderived. Antibodies may have mutations in the CH2 domain of the heavychain that reduce or alter effector function, e.g., Fc receptor bindingand complement activation. For example, antibodies may have mutationssuch as those described in U.S. Pat. Nos. 5,624,821 and 5,648,260.Antibodies may also have mutations that stabilize the disulfide bondbetween the two heavy chains of an immunoglobulin, such as mutations inthe hinge region of IgG4, as disclosed in, e.g., Angal et al. (1993)Mol. Immunol. 30:105-08.

The polypeptides and antibodies of the present invention also encompassproteins that are structurally different from the disclosed polypeptidesand antibodies, e.g., which have an altered sequence but substantiallythe same biochemical properties as the disclosed polypeptides andantibodies, e.g., have changes only in functionally nonessential aminoacids. Such molecules include naturally occurring allelic variants anddeliberately engineered variants containing alterations, substitutions,replacements, insertions, or deletions. Techniques for such alterations,substitutions, replacements, insertions, or deletions are well known tothose skilled in the art.

Antibodies of the invention may additionally be produced usingtransgenic nonhuman animals that are modified so as to produce fullyhuman antibodies rather than the animal's endogenous antibodies inresponse to challenge by an antigen. See, e.g., PCT publication WO94/02602. The endogenous genes encoding the heavy and lightimmunoglobulin chains in the nonhuman host have been incapacitated, andactive loci encoding human heavy and light chain immunoglobulins areinserted into the host's genome. The human genes are incorporated, forexample, using yeast artificial chromosomes containing the requisitehuman DNA segments. An animal which provides all the desiredmodifications is then obtained as progeny by crossbreeding intermediatetransgenic animals containing fewer than the full complement of themodifications. One embodiment of such a nonhuman animal is a mouse, andis termed the XENOMOUSE™ as disclosed in PCT publications WO 96/33735and WO 96/34096. This animal produces B cells that secrete fully humanimmunoglobulins. The antibodies can be obtained directly from the animalafter immunization with an immunogen of interest, as, for example, apreparation of a polyclonal antibody, or alternatively from immortalizedB cells derived from the animal, such as hybridomas producing monoclonalantibodies. Additionally, the genes encoding the immunoglobulins withhuman variable domains can be recovered and expressed to obtain theantibodies directly, or can be further modified to obtain analogs ofantibodies such as, for example, single chain Fv molecules.

Consequently, the term antibody or antigen binding protein as usedherein includes intact antibodies, fragments of antibodies, e.g., Fab,F(ab′)2 Fd, dAb and scFv fragments, and intact antibodies and fragmentsthat have been mutated either in their constant and/or variable domains(e.g., mutations to produce chimeric, partially humanized, or fullyhumanized antibodies, as well as to produce antibodies with a desiredtrait, e.g., enhanced GDF8 binding and/or reduced FcR binding). As suchthese antibodies or antigen binding protein are included in the scope ofthe invention, provided that the antibody or antigen binding proteinspecifically interacts with GDF8.

Other protein-binding molecules may also be employed to modulate theactivity of GDF8. Such antigen binding molecules include small modularimmunopharmaceutical (SMIP™) drugs (Trubion Pharmaceuticals, Seattle,Wash.). SMIPs are single-chain polypeptides composed of a binding domainfor a cognate structure such as an antigen, a counterreceptor or thelike, a hinge-region polypeptide having either one or no cysteineresidues, and immunoglobulin CH2 and CH3 domains. SMIPs and their usesand applications are disclosed in, e.g., U.S. Published Patent Appln.Nos. 2003/0118592, 2003/0133939, 2004/0058445, 2005/0136049,2005/0175614, 2005/0180970, 2005/0186216, 2005/0202012, 2005/0202023,2005/0202028, 2005/0202534, and 2005/0238646, and related patent familymembers thereof, all of which are hereby incorporated by referenceherein in their entireties.

The binding capacity of an antibody or antigen binding protein of theinvention may be measured by the following methods: Biacore analysis,enzyme linked immunosorbent assay (ELISA), X-ray crystallography,sequence analysis and scanning mutagenesis as described in the Examplesbelow, and other methods that are well known in the art. The ability ofan antibody or antigen binding protein of the invention to inhibit,reduce, and/or neutralize one or more GDF8-associated activities may bemeasured by the following nonlimiting list of methods: assays formeasuring the proliferation of a GDF8-dependent cell line; assays formeasuring the expression of GDF8-mediated polypeptides; assays measuringthe activity of downstream signaling molecules; assays testing theefficiency of an antibody or antigen binding protein of the invention toprevent muscle disorders in a relevant animal model; assays as describedin the Examples below; and other assays that are well known in the art.

A further aspect of the invention provides a method of selectingantibodies capable of specifically interacting with GDF8, and/orspecifically antagonizing one or more GDF8 activities. The methodcomprises: contracting a plurality of antibodies with GDF8; choosing asecond plurality of antibodies that bind to GDF8; testing the ability ofthe second plurality of antibodies to bind other members of the TGF-βsuperfamily; and selecting a third plurality of antibodies from thesecond plurality of antibodies wherein the third plurality of antibodiesbinds with less affinity to other members of the TGF-β super family.

In another embodiment, the method further comprises the steps of:testing the ability of the third plurality of antibodies to antagonizeat least one GDF8 activity (e.g., prevent GDF8 from binding to the GDF8receptor); and selecting antibodies capable of antagonizing one or moreGDF8 activity (e.g., preventing GDF8 from binding to its receptor).

The anti-GDF8 antibodies of the invention are also useful for isolating,purifying, and/or detecting GDF8 in supernatant(s), cellular lysates, oron a cell surface. Antibodies disclosed in this invention can be useddiagnostically to monitor GDF8 protein levels as part of a clinicaltesting procedure. Additionally, antibodies of the invention can be usedin treatments requiring the neutralization and/or inhibition of one ormore GDF8-associated disorders, e.g., treatments for muscle-relatedpathologies. The present invention also provides novel isolated andpurified polynucleotides and polypeptides related to novel antibodiesdirected against human GDF8. The genes, polynucleotides, proteins, andpolypeptides of the present invention include, but are not limited to,murine and humanized antibodies to GDF8, e.g., RK22, and variantsthereof.

Antagonist Recombinant Polynucleotides and Polypeptides

The present invention further provides as specific GDF8 antagonists theisolated and purified nucleic acids that encode epitopes specific toGDF8, or the peptide mimetics or antibodies thereto, as described above.Nucleic acids according to the present invention may comprise DNA or RNAand may be wholly or partially synthetic. Reference to a nucleotidesequence as set out herein encompass DNA molecules with the specifiedsequences or genomic equivalents, as well as RNA molecules with thespecified sequences in which T is substituted with U, unless contextrequires otherwise.

The isolated polynucleotides of the present invention, for example, SEQID NOs: 3, 5, 7, 9 11, 13 and 15 may be used as hybridization probes andprimers to identify and isolate nucleic acids having sequences identicalto or similar to those encoding the disclosed polynucleotides. As anonlimiting example, the polynucleotides isolated using antibody orantigen binding protein polynucleotides in this fashion may be used, forexample, to produce specific antibodies against GDF8 or to identifycells expressing such antibodies. Hybridization methods for identifyingand isolating nucleic acids include polymerase chain reaction (PCR),Southern hybridizations, in situ hybridization and Northernhybridization, and are well known to those skilled in the art.

Hybridization reactions can be performed under conditions of differentstringencies. The stringency of a hybridization reaction includes thedifficulty with which any two nucleic acid molecules will hybridize toone another. Preferably, each hybridizing polynucleotide hybridizes toits corresponding polynucleotide under reduced stringency conditions,more preferably stringent conditions, and most preferably highlystringent conditions. Examples of stringency conditions are shown inTable 3 below: highly stringent conditions are those that are at leastas stringent as, for example, conditions A-F; stringent conditions areat least as stringent as, for example, conditions G-L; and reducedstringency conditions are at least as stringent as, for example,conditions M-R.

TABLE 3 Hybrid Hybridization Wash Con- Length Temperature andTemperature and dition Hybrid (bp)1 Buffer2 Buffer2 A DNA:DNA >50 65°C.; 1X SSC -or- 65° C.; 0.3X SSC 42° C.; 1X SSC, 50% formamide B DNA:DNA<50 TB*; 1X SSC TB*; 1X SSC C DNA:RNA >50 67° C.; 1X SSC -or- 67° C.;0.3X SSC 45° C.; 1X SSC, 50% formamide D DNA:RNA <50 TD*; 1X SSC TD*; 1XSSC E RNA:RNA >50 70° C.; 1X SSC -or- 70° C.; 0.3X SSC 50° C.; 1X SSC,50% formamide F RNA:RNA <50 TF*; 1X SSC TF*; 1X SSC G DNA:DNA >50 65°C.; 4X SSC -or- 65° C.; 1X SSC 42° C.; 4X SSC, 50% formamide H DNA:DNA<50 TH*; 4X SSC TH*; 4X SSC I DNA:RNA >50 67° C.; 4X SSC -or- 67° C.; 1XSSC 45° C.; 4X SSC, 50% formamide J DNA:RNA <50 TJ*; 4X SSC TJ*; 4X SSCK RNA:RNA >50 70° C.; 4X SSC -or- 67° C.; 1X SSC 50° C.; 4X SSC, 50%formamide L RNA:RNA <50 TL*; 2X SSC TL*; 2X SSC M DNA:DNA >50 50° C.; 4XSSC -or- 50° C.; 2X SSC 40° C.; 6X SSC, 50% formamide N DNA:DNA <50 TN*;6X SSC TN*; 6X SSC O DNA:RNA >50 55° C.; 4X SSC -or- 55° C.; 2X SSC 42°C.; 6X SSC, 50% formamide P DNA:RNA <50 TP*; 6X SSC TP*; 6X SSC QRNA:RNA >50 60° C.; 4X SSC -or- 60° C.; 2X SSC 45° C.; 6X SSC, 50%formamide R RNA:RNA <50 TR*; 4X SSC TR*; 4X SSC 1The hybrid length isthat anticipated for the hybridized region(s) of the hybridizingpolynucleotides. When hybridizing a polynucleotide to a targetpolynucleotide of unknown sequence, the hybrid length is assumed to bethat of the hybridizing polynucleotide. When polynucleotides of knownsequence are hybridized, the hybrid length can be determined by aligningthe sequences of the polynucleotides and identifying the region orregions of optimal sequence complementarity. 2SSPE (1xSSPE is 0.15MNaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted forSSC (1xSSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridizationand wash buffers; washes are performed for 15 minutes afterhybridization is complete. TB*-TR*: The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be5-10° C. less than the melting temperature (Tm) of the hybrid, where Tmis determined according to the following equations. For hybrids lessthan 18 base pairs in length, Tm (° C.) = 2(# of A + T bases) + 4(# ofG + C bases). For hybrids between 18 and 49 base pairs in length, Tm (°C.) = 81.5 + 16.6(log10Na+) + 0.41(% G + C) − (600/N), where N is thenumber of bases in the hybrid, and Na+ is the concentration of sodiumions in the hybridization buffer (Na+ for 1X SSC = 0.165 M).

Additional examples of stringency conditions for polynucleotidehybridization are provided in Sambrook et al., Molecular Cloning: ALaboratory Manual, Chs. 9 & 11, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989), and Ausubel et al., eds., CurrentProtocols in Molecular Biology, Sects. 2.10 & 6.3-6.4, John Wiley &Sons, Inc. (1995), herein incorporated by reference.

The isolated polynucleotides of the present invention may be used ashybridization probes and primers to identify and isolate DNAs havingsequences encoding allelic variants of the disclosed polynucleotides.Allelic variants are naturally occurring alternative forms of thedisclosed polynucleotides that encode polypeptides that are identical toor have significant similarity to the polypeptides encoded by thedisclosed polynucleotides. Preferably, allelic variants have at least90% sequence identity (more preferably, at least 95% identity; mostpreferably, at least 99% identity) with the disclosed polynucleotides.

The isolated polynucleotides of the present invention may also be usedas hybridization probes and primers to identify and isolate DNAs havingsequences encoding polypeptides homologous to the disclosedpolynucleotides. These homologs are polynucleotides and polypeptidesisolated from a different species than that of the disclosedpolypeptides and polynucleotides, or within the same species, but withsignificant sequence similarity to the disclosed polynucleotides andpolypeptides. Preferably, polynucleotide homologs have at least 50%sequence identity (more preferably, at least 75% identity; mostpreferably, at least 90% identity) with the disclosed polynucleotides,whereas polypeptide homologs have at least 30% sequence identity (morepreferably, at least 45% identity; most preferably, at least 60%identity) with the disclosed antibodies/polypeptides. Preferably,homologs of the disclosed polynucleotides and polypeptides are thoseisolated from mammalian species.

The isolated polynucleotides of the present invention may also be usedas hybridization probes and primers to identify cells and tissues thatexpress the epitope(s) specific to GDF8 or antibodies of the presentinvention and the conditions under which they are expressed.

Additionally, the isolated polynucleotides of the present invention maybe used to alter (i.e., enhance, reduce, or modify) the expression ofthe genes corresponding to the polynucleotides of the present inventionin a cell or organism. These “corresponding genes” are the genomic DNAsequences of the present invention that are transcribed to produce themRNAs from which the polynucleotides of the present invention arederived.

Altered expression of sequences related to the invention in a cell ororganism may be achieved through the use of various inhibitorypolynucleotides, such as antisense polynucleotides, ribozymes that bindand/or cleave the mRNA transcribed from the genes of the invention,triplex-forming oligonucleotides that target regulatory regions of thegenes, and short interfering RNA that causes sequence-specificdegradation of target mRNA (e.g., Galderisi et al. (1999) J. Cell.Physiol. 181:251-57; Sioud (2001) Curr. Mol. Med. 1:575-88; Knauert andGlazer (2001) Hum. Mol. Genet. 10:2243-51; Bass (2001) Nature411:428-29). Such inhibitory polynucleotides are considered antagonistsof the invention. A skilled artisan will recognize that inhibitorypolynucleotides of the invention should be directed against theepitope(s) specific to GDF8 as provided above (and not antagonistantibodies of the invention).

The inhibitory triplex-forming oligonucleotides (TFOs) encompassed bythe present invention bind in the major groove of duplex DNA with highspecificity and affinity (Knauert and Glazer, supra). Expression of thegenes of the present invention can be inhibited by targeting TFOscomplementary to the regulatory regions of the genes (i.e., the promoterand/or enhancer sequences) to form triple helical structures thatprevent transcription of the genes.

In one embodiment of the invention, the inhibitory polynucleotides ofthe present invention are short interfering RNA (siRNA) molecules (see,e.g., Galderisi et al. (1999) J. Cell Physiol. 181:251-57; Sioud (2001)Curr. Mol. Med. 1:575-88). These siRNA molecules are short duplex RNAmolecules that cause sequence-specific degradation of the targeted mRNA.This degradation is known as RNA interference (RNAi) (e.g., Bass (2001)Nature 411:428-29). Originally identified in lower organisms, RNAi hasbeen effectively applied to mammalian cells and has recently been shownto prevent fulminant hepatitis in mice treated with siRNA moleculestargeted to Fas mRNA (Song et al. (2003) Nature Med. 9:347-51). Inaddition, intrathecally delivered siRNA has recently been reported toblock pain responses in two models (agonist-induced pain model andneuropathic pain model) in the rat (Dorn et al. (2004) Nucleic AcidsRes. 32(5):e49).

The duplex structure of siRNA molecules of the invention may compriseone or more strands of polymerized RNA, i.e., the duplex structure maybe formed by a single-self complementary RNA strand comprising a hairpinloop or two complementary strands. siRNA sequences with insertions,deletions, and single point mutations relative to the targeted sequencehave also been found to be effective in inhibiting the expression of thetargeted sequence (Fire et al., U.S. Pat. No. 6,506,559). Accordingly,it is preferred that siRNA molecules of the invention comprise anucleotide sequence with substantial sequence identity to at least aportion of the mRNA corresponding to a targeted epitope specific to GDF8of the invention. For example, the duplex region of an siRNA molecule ofthe invention may have greater than 90%, sequence identity, andpreferably 100% sequence identity, to at least of portion of the mRNAcorresponding to the targeted epitope specific to GDF8. Alternatively,substantial sequence identity may be defined as the ability of at leastone strand of the duplex region of the siRNA molecule to hybridize to atleast a portion of the targeted epitope specific to GDF8 under at least,e.g., stringent conditions as defined as conditions G-L in Table 3,above. In a preferred, but nonlimiting, embodiment of the invention, thesiRNA molecule hybridizes to at least of a portion of the targetedepitope specific to GDF8 under highly stringent conditions, e.g., thosethat are at least as stringent as, for example, conditions A-F in Table3, above. The length of the substantially identical nucleotide sequencesmay be at least 10, 15, 19, 21, 23, 25, 50, 100, 200, 300, 400, or 500nucleotides, is preferably 19-27 nucleotides, and is most preferably 19or 21 nucleotides (see Fire, et al., supra).

The inhibitory polynucleotides of the invention may be designed based oncriteria well known in the art (e.g., Elbashir et al. (2001) EMBO J.20:6877-88) and/or by using well-known algorithms (e.g., publiclyavailable algorithms). For example, the targeting portion of aninhibitory polynucleotide of the invention (e.g., the duplex region ofan siRNA molecule) preferably should begin with AA (most preferred), TA,GA, or CA; an siRNA molecule of the invention preferably should comprisea sequence whereby the GC ratio is 45-55%; an siRNA molecule of theinvention preferably should not contain three of the same nucleotides ina row; and an siRNA molecule of the invention preferably should notcontain seven mixed G/Cs in a row. Based on these criteria, or on otherknown criteria (e.g., Reynolds et al. (2004) Nat. Biotechnol.22:326-30), siRNA molecules of the present invention that target anepitope specific to GDF8 may be designed by one of ordinary skill in theart. For example, in one embodiment, an siRNA molecule of the inventionmay have and/or consist essentially of a nucleotide sequence selectedfrom the group consisting of the nucleotide sequence of SEQ ID NO:3, thenucleotide sequence of SEQ ID NO:5, the nucleotide sequence of SEQ IDNO:7, the nucleotide sequence of SEQ ID NO:9, the nucleotide sequence ofSEQ ID NO:11, and fragments thereof. In this embodiment, an siRNAmolecule of the invention further comprises the complement of thenucleotide sequence of SEQ ID NO:3, the complement of the nucleotidesequence of SEQ ID NO:5, the complement of the nucleotide sequence ofSEQ ID NO:7, the complement of the nucleotide sequence of SEQ ID NO:9,the complement of the nucleotide sequence of SEQ ID NO:11, and thecomplements of fragments thereof.

For example, the siRNA molecules of the present invention may begenerated by annealing two complementary single-stranded RNA moleculestogether (Fire et al., supra) or through the use of a single hairpin RNAmolecule that folds back on itself to produce the requisitedouble-stranded portion (Yu et al. (2002) Proc. Natl. Acad. Sci. USA99:6047-52). The siRNA molecules may be chemically synthesized (Elbashiret al. (2001) Nature 411:494-98) or produced by in vitro transcriptionusing single-stranded DNA templates (Yu et al., supra). Alternatively,the siRNA molecules can be produced biologically, either transiently (Yuet al., supra; Sui et al. (2002) Proc. Natl. Acad. Sci. USA 99:5515-20)or stably (Paddison et al. (2002) Proc. Natl. Acad. Sci. USA99:1443-48), using an expression vector(s), described below, comprisingpolynucleotides related to the present invention in sense and/orantisense orientation relative to their promoter. Recombinant RNApolymerase may be used for transcription in vivo or in vitro, orendogenous RNA polymerase of a modified cell may mediate transcriptionin vivo. Recently, reduction of levels of target mRNA in primary humancells, in an efficient and sequence-specific manner, was demonstratedusing adenoviral vectors that express hairpin RNAs, which are furtherprocessed into siRNA molecules (Arts et al. (2003) Genome Res.13:2325-32).

The inhibitory polynucleotides of the invention may be constructed usingchemical synthesis and enzymatic ligation reactions including procedureswell known in the art. The nucleoside linkages of chemically synthesizedpolynucleotides may be modified to enhance their ability to resistnuclease-mediated degradation, avoid a general panic response in someorganisms that is generated by duplex RNA, and/or to increase theirsequence specificity. Such linkage modifications include, but are notlimited to, phosphorothioate, methylphosphonate, phosphoroamidate,boranophosphate, morpholino, and peptide nucleic acid (PNA) linkages(Galderisi et al., supra; Heasman (2002) Dev. Biol. 243:209-14;Micklefield (2001) Curr. Med. Chem. 8:1157-79).

As described above, the isolated polynucleotides, or continuous portionsthereof, related to the present invention may be operably linked insense or antisense orientation to an expression control sequence and/orligated into an expression vector for recombinant expression of theinhibitory polynucleotides (e.g., siRNA molecules) of the invention.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone nucleic acid of the invention as above.

The isolated polynucleotides of the present invention may be operablylinked to an expression control sequence for recombinant production ofthe specific epitopes (e.g., as peptide mimetics) or antibodies of thepresent invention. Additionally one of skill in the art will recognizethat the antibody or antigen binding protein encoding polynucleotides ofthe invention may be operably linked to well-known nucleotide sequencesencoding the constant domain for various antibody isotypes. For example,a polynucleotide of the invention that encodes a light chain variabledomain of the invention (e.g., polynucleotides with a nucleotidesequence set forth as SEQ ID NO: 15 may be operably linked to anucleotide sequence that encodes the constant domain (or derivativesthereof) of either a K light chain or A light chain, such that theexpression of the linked nucleotides will result in a full kappa orlambda light chain with a variable domain that specifically interactswith and/or specifically antagonizes GDF8. Similarly, a polynucleotideof the invention that encodes a heavy chain variable domain of theinvention (e.g., a polynucleotide with a nucleotide sequence set forthas SEQ ID NOs: 13 may be operably linked to a nucleotide sequence thatencodes the constant domain of a heavy chain isotype (or derivativesthereof), e.g., IgM, IgD, IgE, IgG and IgA. General methods ofexpressing recombinant proteins are well known in the art. Suchrecombinant proteins may be expressed in soluble form for use intreatment of disorders related to GDF8 The recombinant expressionvectors of the invention may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication), tag sequences such as histidine, and selectablemarker genes. The selectable marker gene facilitates selection of hostcells into which the vector has been introduced. For example, typicallythe selectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

Suitable vectors, containing appropriate regulatory sequences, includingpromoter sequences, terminator sequences, polyadenylation sequences,enhancer sequences, marker genes and other sequences as appropriate, maybe either chosen or constructed. Vectors may be plasmids or viral, e.g.,phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd ed., Sambrook etal., Cold Spring Harbor Laboratory Press, 1989. Many known techniquesand protocols for manipulation of nucleic acid, for example, inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, 2nd ed., Ausubel et al. eds., John Wiley & Sons, 1992.

The present invention also provides a host cell that comprises one ormore constructs as above, e.g., a recombinant nucleic acid encoding anyepitope specific to GDF8, CDR (H1, H2, H3, L1, L2, or L3), VH domain, VLdomain, or specific antigen-binding fragment as provided herein, formsan aspect of the present invention.

The present invention also includes a method of producing a peptide byexpressing the protein from the encoding nucleic acid in a host cell.Expression may be achieved by culturing recombinant host cellscontaining the nucleic acid under appropriate conditions.

A number of cell lines are suitable host cells for recombinantexpression of the polypeptides and antibodies of the present invention.Mammalian host cell lines include but are not limited to: COS cells, CHOcells, 293T cells, A431 cells, 3T3 cells, CV-1 cells, HeLa cells, Lcells, BHK21 cells, HL-60 cells, U937 cells, HaK cells, Jurkat cells aswell as cell strains derived from in vitro culture of primary tissue andprimary explants. Such host cells also allow splicing of thepolynucleotides of the invention that consist of genomic DNA.

Alternatively, it may be possible to recombinantly produce thepolypeptides and antibodies of the present invention in lower eukaryotessuch as yeast or in prokaryotes. Potentially suitable yeast strainsinclude but are not limited to Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, and Candida strains.Potentially suitable bacterial strains include Escherichia coli,Bacillus subtilis, and Salmonella typhimurium. If the polypeptides ofthe present invention are made in yeast or bacteria, it may be necessaryto modify them by, for example, phosphorylation or glycosylation ofappropriate sites, in order to obtain functional proteins. Such covalentattachments may be accomplished using well-known chemical or enzymaticmethods.

The polypeptides and antibodies of the present invention may also berecombinantly produced by operably linking the isolated polynucleotidesof the present invention to suitable control sequences in one or moreinsect expression vectors, such as baculovirus vectors, and employing aninsect cell expression system. Materials and methods for baculovirus/Sf9expression systems are commercially available in kit form (e.g., theMAXBAC® kit, Invitrogen, Carlsbad, Calif.).

Following recombinant expression in the appropriate host cells, thepolypeptides and antibodies of the present invention may be purifiedfrom culture medium or cell extracts using known purification processes,such as gel filtration and ion exchange chromatography. Purification mayalso include affinity chromatography with agents known to bind thepolypeptides and antibodies of the present invention. These purificationprocesses may also be used to purify the polypeptides and antibodies ofthe present invention from natural sources.

Alternatively, the polypeptides and antibodies of the present inventionmay be recombinantly expressed in a form that facilitates purification.For example, the polypeptides may be expressed as fusions with proteinssuch as maltose-binding protein (MBP), glutathione-S-transferase (GST),or thioredoxin (TRX). Kits for expression and purification of suchfusion proteins are commercially available from New England BioLabs(Beverly, Mass.), Pharmacia (Piscataway, N.J.), and Invitrogen,respectively. The polypeptides and antibodies of the present inventioncan also be tagged with a small epitope and subsequently identified orpurified using a specific antibody or antigen binding protein to theepitope. A preferred epitope is the FLAG epitope, which is commerciallyavailable from Eastman Kodak (New Haven, Conn.).

The polypeptides and antibodies of the present invention may also beproduced by known conventional chemical synthesis. Methods forchemically synthesizing the polypeptides and antibodies of the presentinvention are well known to those skilled in the art. Such chemicallysynthetic polypeptides and antibodies may possess biological propertiesin common with the natural purified polypeptides and antibodies, andthus may be employed as biologically active or immunological substitutesfor the natural polypeptides and antibodies.

A further aspect of the present invention provides a host cellcomprising nucleic acids, polypeptides, vectors, or antibodies andfragments thereof as disclosed herein. A still further aspect provides amethod comprising introducing a nucleic acid of the invention into ahost cell. The introduction may employ any available technique. Foreukaryotic cells, suitable techniques may include calcium phosphatetransfection, DEAE Dextran, electroporation, liposome-mediatedtransfection and transduction using a retrovirus or another virus, e.g.,vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and infection using bacteriophage.

The introduction of nucleic acids may be followed by causing or allowingprotein production from the nucleic acid, e.g., by culturing the hostcells under conditions suitable for gene expression. Such conditions arewell-known in the art.

Inhibitory polynucleotides, epitope(s) specific to GDF8 (e.g., aspeptide mimetics and/or immunogens), specific antibody or antigenbinding protein fragments, VH domains, and/or VL domains, and encodingnucleic acid molecules and vectors according to the present inventionmay be provided isolated and purified, e.g., from their naturalenvironment, in substantially pure or homogeneous form, or, in the caseof nucleic acids, free or substantially free of nucleic acids or genesof origin other than the sequence encoding a polypeptide with therequired function.

Methods to Detect and Quantify GDF8 in Biological Samples

The present invention relates to methods to detect and quantify GDF-8 inbiological samples. In some embodiments, the methods compriseimmunoassays to detect and quantify both free and total GDF-8 in serum,blood, and plasma. In one instance, the immunoassays provide data thatare useful as biomarkers of anti-GDF-8 therapies. Specifically, thedisclosed immunoassays may be useful as predictive/prognostic markers ofclinical outcome at baseline prior to anti-GDF-8 therapy, as a marker ofexposure to anti-GDF-8 therapies, as a marker of anti-GDF-8 drugefficacy or response, and as a diagnostic marker of GDF-8 involvement ina particular disease state or biological process.

In particular, the methods provide diagnostic and/or prognostic methodsfor detecting, diagnosing, and predicting a GDF-8 associated disease ordisorder in mammals with or at risk for developing a GDF-8 associateddisease or disorder. The methods are especially suitable for use inevaluating the suitability of human patients to receive GDF-8 modulatingagents, for example, those that bind to GDF-8, or inhibit a biologicalactivity of GDF-8.

In certain embodiments, the invention provides methods to monitor theprogress of individuals who are receiving GDF-8 modulating agents oranti-GDF-8 therapies. For example, methods are provided to assess anindividual's response to therapy with a GDF-8 modulating agent. In orderto assess an individual's response to therapy, the immunoassay methodsmay be provided prior to, during, and post administration of the GDF-8modulating agent. Methods to detect the presence of GDF-8 in mammalsthat are receiving the therapeutic antibody MYO-029 are also encompassedby the invention.

In one embodiment, the immunoassay methods of the invention detect freeGDF-8. For example, free GDF-8 is GDF-8 that is not bound to GDF-8binding proteins or GDF-8 modulating agents, such as, GDF-8 binding orneutralizing antibodies. In another embodiment, methods to detect totalGDF-8, for example, free GDF-8 plus any bound GDF-8, are encompassed.

An individual having, or at risk for developing, a muscle-relateddisorder is a candidate for the methods provided herein. Inhibition ofGDF-8 activity increases muscle mass in individuals, including thosesuffering from muscle-related disorders. A number of disorders areassociated with functionally impaired muscle tissue, e.g., musculardystrophies, amyotrophic lateral sclerosis (ALS), muscle atrophy, organatrophy, frailty, congestive obstructive pulmonary disease, heartfailure, sarcopenia, cachexia, and muscle wasting syndromes caused byother diseases and conditions. Further, an individual or mammal desiringto increase muscle mass or muscle strength, to increase growth or muscletissue mass in feedstock animals, is a candidate for a method providedherein.

An individual having, or at risk for developing, an adipose tissue,metabolic, or bone-related disorder or condition is also a candidate fora method as described and claimed herein. Such disorders or conditionsinclude those associated with glucose homeostasis such as, e.g.,development of type 2 diabetes, impaired glucose tolerance, metabolicsyndromes (e.g., syndrome X), insulin resistance induced by trauma, suchas burns or nitrogen imbalance, and adipose tissue disorders (e.g.,obesity) (Kim et al., Biochem. Biophys. Res. Comm. 281:902-906 (2001)).For example, GDF-8 modulates preadipocyte differentiation to adipocytes(Id.) and inhibits adipocyte formation from mesenchymal precursor cellsand preadipocytes (Rebbapragada et al., Mol. Cell. Bio. 23:7230-7242(2003)). Fat accumulation is reduced both in GDF-8 knockout mice and inwild-type adult mice in which GDF-8 protein has been systematicallyadministered (McPherron et al., J. Clinical Invest. 109:595-601 (2002);Zimmers et al., Science 296:1486-1488 (2002)).

Other uses for the methods of the present invention will be apparent tothose of skill in the art, and are further exemplified below.

Immunoassays

The immunoassays described herein are sandwich-type ELISA's that utilizeat least two anti-GDF-8 antibodies; one present as a GDF-8 capturereagent specific for GDF8 and one present as a GDF-8 detection reagentspecific for GDF8. Both antibodies are capable of binding GDF-8 antigenspresent in biological samples. One of the antibodies preferentiallyrecognizes GDF-8 over BMP-11. Both antibodies are capable of recognizingand binding GDF-8. Furthermore, in certain embodiments, the antibodiesare capable of binding to GDF-8 that is present in any of its biologicalforms (e.g., active GDF-8, latent GDF-8, GDF-8 bound to serum proteins,GDF-8 bound to neutralizing anti-GDF-8 antibodies MYO-029).

In certain embodiments, the antibody used in the subject assay is RK35(see SEQ ID NO:s 31-35 and US Application US2007/0087000 herebyincorporated by reference), which is an isolated murine monoclonalantibody that binds to GDF-8. In some embodiments, RK35 is utilized as acapture antibody. Fragments of RK35 that bind to GDF-8 may also be usedin the methods of the invention.

In certain embodiments, a second antibody used in the subject assay isRK22, an isolated murine monoclonal antibody that binds to GDF-8. RK22does not bind to BMP-11, as exemplified below. In some embodiments, RK22is utilized as a detection reagent, in some embodiments it is used as acapture reagent. Fragments of RK22 that bind to GDF-8 may also be usedin the assays of the invention.

In another embodiment, the immunoassays of the present invention utilizethe antibody MYO-029, which is a human IgG1 anti-GDF-8 antibody. MYO-029(see SEQ ID NO:s 33 and 34, US Published Applications 2006/0240488 and2006/0240487, hereby incorporated by reference) may be used to block thedetection of GDF-8 in the immunoassays so as to obtain a backgroundlevel that can be subtracted from the signal generated in the absence ofMYO-029. This embodiment can be employed to increase the sensitivity andaccuracy of the quantitative assay.

The antibodies useful in the methods of the invention also encompassantigen-binding fragments, such as, for example, Fv fragments, whichconsist of the VH and VL domains, Fab fragments (Fragment antigenbinding), which consist of the VH—CH1 and VL-CL domains covalentlylinked by a disulfide bond between the constant regions. For otherpossible antigen binding fragments, and a review of the antibodystructure, see Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, eds. Harlow et al., 1988.

E. coli cultures individually transformed with the phagemid vectorpCANTAB6 encoding nongermlined scFv's MYO-029 was deposited on Oct. 2,2002, at American Tissue Culture Collection (ATCC) under respectiveDeposit Designation Numbers PTA-4741. The address of the depository is10801 University Blvd, Manassas, Va. 20110, U.S.A.

After combining the sample containing GDF8 with the capture reagent anynon-bound components are removed by washing, and components are thencontacted with a sample suspected of containing GDF-8 under suitablebinding conditions. After washing to remove any non-bound molecules, asecond anti-GDF-8 antibody is added under suitable binding conditions.This second antibody is termed the detection antibody or reagent. Thedetection antibody may include a detectable label, and bind moleculesthat have reacted with the capture antibody. Thus, any GDF-8 presentwill bind both the capture reagent bound to GDF8 in the sample, as wellas the detection antibody reagent. Unbound molecules and components areremoved by washing. The presence of a label therefore indicates thepresence of GDF-8 in the biological sample.

More particularly, a sandwich ELISA method can be used. A biologicalsample containing or suspected of containing GDF-8 is a capture reagentspecific for GDF8. After a period of incubation sufficient to allowGDF-8 binding to the capture reagent, the plate(s) can be washed toremove unbound components and a detection component is added. Thesemolecules are allowed to react with any captured sample GDF-8, the platewashed and the presence of the label detected using methods well knownin the art.

The above-described assay reagents, including the immunoassay withantigens, as well as antibodies to be reacted with the captured sample,may be provided in kits, with suitable instructions and other necessaryreagents, in order to conduct immunoassays as described above. The kitmay also contain, depending on the particular immunoassay used, suitablelabels and other packaged reagents and materials (i.e. wash buffers andthe like). Immunoassays, such as those described above, can be conductedusing these kits.

SPECIFIC EMBODIMENTS OF THE IMMUNOASSAYS Analysis of Free GDF-8

In one embodiment, the present invention comprises a method fordetecting the presence of free GDF-8 in a biological sample. Arepresentative example of this embodiment is depicted in FIG. 21.

As used herein, the term free GDF-8 includes GDF-8 that is present inits active, mature state. Mature GDF-8 may be a monomer, dimer, orhomodimer. Free GDF-8 does not encompass latent GDF-8 (i.e., matureGDF-8 associated with GDF-8 propeptide), GDF-8 associated with GDF-8binding proteins, or GDF-8 that is associated with anti-GDF-8 modulatingagents, such as, for example, GDF-8 binding and neutralizing antibodies.

In one embodiment, methods to detect and quantify free GDF-8 comprisethe following steps: (a) combining a GDF-8 capture antibody and a sampleunder conditions which allow GDF-8, when present in the biologicalsample, to bind to the one or more capture antibodies forming a captureantibody-GDF-8 complex; adding a detection antibody undercomplex-forming conditions, wherein the detection antibody binds thecapture antibody-GDF-8 complex; and (b) detecting complexes formedbetween the capture antibody-GDF-8 complex and the detection antibody,if any, as an indication of GDF-8 in the biological sample.

The detection antibody or antigen binding protein may further comprise adetectable label. In some instances, the detection antibody is notlabeled and a detection agent that specifically recognizes the detectionantibody is utilized.

Analysis of Total GDF-8

In one embodiment, the present invention comprises a method fordetecting the presence of total GDF-8 in a biological sample.

As used herein, the term total GDF-8 includes GDF-8 that is present inits active, mature state, and any GDF-8 that is present in its latentform (i.e., mature GDF-8 associated with GDF-8 propeptide), GDF-8associated with GDF-8 binding proteins, or GDF-8 that is associated withanti-GDF-8 modulating agents, such as, for example, GDF-8 binding andneutralizing antibodies. A measurement of total GDF-8 includes ameasurement of GDF-8 that is bound by therapeutic antibody MYO-029.

Acid Dissociation

In one embodiment, methods to detect and quantify total GDF-8 utilize anacid dissociation method, and comprise the following steps: (a)combining a GDF-8 capture antibody with a biological sample under acidicconditions (between about pH1.0 to about pH 6.0, preferable about pH2.5) which allow GDF-8, when present in the biological sample, to bindto one or more capture antibodies forming a capture antibody-GDF-8complex; adding a GDF-8 detection antibody) under complex-formingconditions, wherein the detection antibody binds the captureantibody-GDF-8 complex; and (b) detecting complexes formed between thecapture antibody-GDF-8 complex and the detection antibody, if any, as anindication of GDF-8 in the biological sample.

The detection antibody or antigen binding protein may further comprise adetectable label. In some instances, the detection antibody is notlabeled and a detection agent that specifically recognizes the detectionantibody is utilized.

Heat Dissociation

In another embodiment, methods to detect and quantify total GDF-8utilize a heat dissociation method, and comprise the following steps:(a) contacting a GDF-8 capture antibody or antigen binding protein witha surface of a solid support; (b) heating a biological sample to atleast 63° C., such as, e.g., 65° C., 70° C., 75° C., 80° C., 85° C., or90° C., for at least 3 minutes, such as, e.g., 5, 7, 9, 10, 12, 14, or15 minutes and combining a biological sample with the solid supportunder conditions which allow GDF-8, when present in the biologicalsample, to bind to one or more capture antibodies forming a captureantibody-GDF-8 complex; (c) adding a detection antibody to the solidsupport from step (b) under complex-forming acidic conditions, whereinthe detection antibody binds the capture antibody-GDF-8 complex; and (d)detecting complexes formed between the capture antibody-GDF-8 complexand the detection antibody, if any, as an indication of GDF-8 in thebiological sample.

The detection antibody or antigen binding protein may further comprise adetectable label. In some instances, the detection antibody is notlabeled and a detection agent that specifically recognizes the detectionantibody is utilized.

One embodiments of this method are depicted in FIG. 12, where it isshown that the antibody MYO-029 inhibits signal produced by thedetection antibody (e.g., biotinylated-RK22). In this manner, the assaybackground can be calculated and subtracted from the value obtained instep (d).

ALTERNATIVE EMBODIMENTS FOR THE ANALYSIS OF FREE AND TOTAL GDF-8

In certain embodiments, the capture antibody is contacted with thesurface of a solid support, or a reaction vessel, for example by beingeither covalently or non-covalently bound to the surface. The contactmay be direct or indirect. The surface may be modified, for example bychemical or radiation treatment to affect the binding characteristics ofthe surface.

In certain embodiments, after contacting the capture antibody with thebiological sample and washed to remove unbound components. Non-specificinteractions may be minimized with a blocking step, wherein a buffercomprising at least one blocking agent, such as a protein that does notspecifically bind to the target is added to the reaction vessel.Blocking buffers may comprise commercially available blocking buffers,serum, bovine serum albumin, milk, casein, gelatin, and/or non-ionicdetergents, for example. In some embodiments the reaction vessel iswashed with a buffer with a pH between about 5 and about 9, such ascitrate buffer, phosphate buffer, Tris buffer or acetate buffer.Alternatively, the buffer is between about pH 3.0 and pH 5.0, forexample, in the acid dissociation methods to detect and quantify totalGDF-8.

The biological sample to be tested in the methods of the invention maybe chosen from serum, blood, plasma, biopsy sample, tissue sample, cellsuspension, saliva, oral fluid, cerebrospinal fluid, amniotic fluid,milk, colostrum, mammary gland secretion, lymph, urine, sweat, synovialfluid, and lacrimal fluid. In certain embodiments, the biological sampleis a fluid. In some embodiments, the biological sample is chosen fromblood, serum, and plasma. In specific embodiments, the biological sampleis serum from, e.g., such as human, monkey, rat, mouse, bovine, ovine,or chicken serum.

In other embodiments, the biological sample is isolated from anindividual or individuals and optionally treated prior to testing. Forexample, the sample may be diluted. The dilution buffer may optionallycomprise a constant amount of a control biological sample, chosen tocorrespond to the test biological sample, for example to control forbackground effects or interference of the sample matrix. In oneembodiment, a test sample of human plasma is diluted in THST (50 mMTris-HCl, pH 8.0, containing 1.0 mM glycine, 0.5 M NaCl, and 0.05% (v/v)Tween 20®) buffer 1:8 fold, and dilutions of the biological samplebeyond 8-fold are prepared in THST plus 12.5% human serum that has beendepleted of GDF-8. A biological sample may be diluted approximately 2,4, 5, 8, 10, 12, 14, 15, 16, 32, 64, or 128-fold. In other embodiments,a biological sample is serially diluted 1:1.5 or 1:1.6 to obtain a rangeof data points that allow verification of dilutional linearity andmatrix effects. For some biological sample matrices, a dilution may beselected at which matrix interference and assay sensitivity areoptimized.

The diluent is not particularly restricted but may comprise serum,including e.g., human serum, human serum that has been depleted ofGDF-8, mouse serum, mouse serum that has been depleted of GDF-8,deionized water or various buffers having a buffer action within therange of pH about 3.0 to pH about 9.0, depending on whether the assay isto be performed at acidic conditions or not. For analysis of free GDF-8,performed at neutral pH, the pH is about 6.5 to about 8.5, about 6.5 toabout 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, or about 8.0to about 8.5 (e.g. citrate buffer, phosphate buffer, Tris buffer,acetate buffer, or borate buffer). For analysis of total GDF-8,performed at an acidic pH, the pH is, for example, about 1.0 to about2.5, about 2.5 to about 5.5, about 2.5 to about 3.0, about 3.0 to about3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, or about 4.5 toabout 5.5, about 5.5 to about 6.5.

In some embodiments, the biological sample may be optionallyfractionated or concentrated using well known methods and then added toan assay as described herein to detect GDF-8. Fractionation (includingpurification) or concentration may be used, for example, if matrixinterference limits detection of a GDF-8 modulating agent in the assay.Fractionation and concentration techniques, include, but are not limitedto, centrifugation, ammonium sulfate precipitation, polyethylene glycolprecipitation, trichloroacetic acid (TCA) precipitation, affinitytechniques (such as immunoprecipitation with a resin conjugated to aspecific binding partner such as an antibody, e.g., an anti-GDF-8antibody), chromatographic techniques, and other separation techniques.

A biological sample may be collected from a naïve individual, or abiological sample may be taken before, during or after administration ofa GDF-8 modulating agent. For example, a sample may be obtained from anindividual 1, 2, 4, 6, 7, 8, 10, 12, 14, 15, 20, 25, 30, or more daysafter administration of a GDF-8 modulating agent. A biological samplemay also be obtained 1, 2, 3, 4, 6, 7, 8, 10, 12, 14, 16, or more weeksafter administration of a GDF-8 modulating agent. In some cases,timepoints of up to a year or beyond are appropriate. Biological samplesmay be tested for both free and total GDF-8. An analysis of total GDF-8is particularly important in individuals being treated with GDF-8modulating agents, as these agents may interfere with the ability ofeither the detection or the capture agent to bind GDF-8 in a biologicalsample. Thus, the acid or heat dissociation method utilized in theanalysis of total GDF-8 becomes particularly important.

In certain embodiments, an aliquot of the biological sample to be testedis contacted with the capture antibody or antigen binding protein andincubated for a period of time sufficient (e.g., 2-120 minutes, or 1-4hours) and under suitable conditions (e.g., 23° C.) to allow binding thecapture antibody to the GDF-8, if any, present in the biological sampleand to allow antibody/GDF-8 complexes to form. In other embodiments, theGDF-8/antibody reaction is conducted under the conditions in routine usefor conventional immunoassays. A typical procedure comprises incubatingor allowing to stand a reaction system comprising the capture antibodyand biological sample at a temperature of not over 45° C., such as,e.g., between about 4° C. and about 40° C., or between about 23° C. andabout 40° C. for between about 0.5 and 40 hours, such as, e.g., betweenabout 1 and about 20 hours.

Following an incubation period, the antibody/GDF-8 complex is, in someembodiments, washed with buffer to remove unbound solutes. In otherembodiments a simultaneous assay is performed, whereby the biologicalsample and detection antibody are added to the reaction vesselconcurrently.

In particular embodiments, in which the detection antibody is addedafter the biological sample, a procedure may comprise incubating orallowing to stand a reaction system comprising the antibody/GDF-8complex and detection antibody at a temperature of not over 45° C., suchas, e.g., between about 4° C. and about 40° C., or between about 25° C.and about 40° C. for between about 0.5 and 40 hours, or between about 1and about 20 hours.

In some embodiments, the detection antibody, antigen binding protein orfragment thereof comprises a detectable label. In further embodiments,the detection antibody is indirectly detected, for example by adetection agent. In some embodiments, the detection agent is in excess,so that essentially all detection antibodies that are present in thereaction vessel are bound.

In some embodiments, a “direct” label may be any molecule bound orconjugated to a specific binding member that is capable of spontaneouslyproducing a detectible signal without the addition of ancillaryreagents. Some examples include a radioisotope (e.g., 125I, 3H, 14C), afluorophore (e.g., luciferase, green fluorescent protein, fluoresceinisothiocyanate, tetramethylrhodamine isothiocyanate,1-N-(2,2,6,6-tetramethyl-1-oxyl-4-piperidyl)-5-N-(aspartate)-2,4-dinitrobenzene),a dye (e.g., phycocyanin, phycoerythrin, Texas Red, o-phthalaldehyde),luminescent molecules, including chemiluminescent and bioluminescentmolecules, colloidal gold particles, colloidal silver particles, othercolloidal metal particles, polystyrene dye particles, minute coloredparticles such as dye sols, and colored latex particles. Many othersuitable label molecules are well known to those skilled in the art andmay be utilized in the methods of the invention.

In certain instances, the label may be an enzyme such as, e.g., alkalinephosphatase, horseradish peroxidase, glucose oxidase, orβ-galactosidase. In various embodiments, the substrates to be used withthe specific enzymes are chosen for the production, in the presence ofthe corresponding enzyme, of a detectable change in color, fluorescence,or luminescence. The enzyme may be conjugated to the antibody or antigenbinding protein by glutaraldehyde or reductive amination cross-linking.As will be readily recognized, however, wide varieties of differentconjugation techniques exist, and are readily available to the skilledartisan.

In a preferred embodiment, the detection antibody or antigen bindingprotein is biotinylated. Anti-GDF-8 antibodies useful as detectionantibodies may be biotinylated as set forth in Example 1 (see, e.g.,Example 1: section 1, subsection 5). Various biotinylation reagents arecapable of efficiently labeling proteins, including antibodies. Molarratios of biotin derivative to antibody may be about 10, 15, 20, 40, or80 to 1, and reaction times, reactant concentrations, and temperaturesmay be varied to adjust the amount of biotin incorporated in thereaction. Biotin derivatives are well known and available in the art,including variable spacer arms, modifications to affect solubility,and/or reactive groups to allow cleavage of the biotin moiety.Succinimidyl esters of biotin and its derivatives, includingwater-soluble sulfosuccinimidyl esters may be used for biotinylation ofGDF-8, for example. To quantitate the amount of biotin incorporated,well-known analytical and sizing techniques are used including, forexample, reverse phase high pressure liquid chromatography, massspectroscopy, etc. Additionally, commercial kits for quantitating biotinby colorimetric or fluorimetric assays, for example, are available (see,e.g., EZ™ Biotin Quantitation Kit, Pierce, utilizing HABA(2-(4′-hydroxyazo benzene)-benzoic acid)).

In one embodiment, biotinylated RK-22 is a detection agent for detectingGDF-8 binding to RK35.

In a particular embodiment, the biotinylated and/or enzyme-labeleddetection agent such as an antibody or antigen binding protein is addedto the GDF-8/antibody complex, and allowed to bind. The excess reagentis washed away, and a solution containing an appropriate substrate isthen added to reaction vessel. The substrate undergoes anenzyme-catalyzed reaction resulting in aspectrophotometrically-measurable change that is indicative of theamount of GDF-8 present in the sample.

For example, a biotinylated detection antibody or antigen bindingprotein can be detected through its interaction with an avidin-enzymeconjugate, e.g., avidin-horseradish peroxidase, after sequentialincubation with the avidin-enzyme conjugate and a suitable chromogenicor fluorogenic substrate. A biotinylated detection antibody may also bedetected with Europium labeled streptavidin.

In certain embodiments, an antibody/GDF-8/antibody complex associatedwith the surface of the reaction vessel is detected by qualitative orquantitative assessment of the signal of the label. In some instances,the label is measured directly, e.g., by fluorescence or luminescence,or indirectly, via addition of a substrate. In others, the label ismeasured following incubation with an additional reagent. In embodimentsin which the label is biotin, an avidin conjugate (such as horseradishperoxidase) may be added in a subsequent step. In one particularembodiment, the avidin conjugate may bind to the immobilized detectionantibody or antigen binding protein. Excess avidin conjugate is washedaway. A substrate of the enzyme is then added, resulting in a measurablechange in, e.g., color, fluorescence, or luminescence. In someembodiments the substrate for horseradish peroxidase is3,3′,5,5′-tetramethylbenzidine.

Quantitation of Free and Total GDF-8

GDF-8 levels may be quantified using methods well known to those ofskill in the art. In certain embodiments, the GDF-8 levels in abiological sample are compared to a known level, such as is obtained,for example, by using a standard curve. The generation of GDF-8 standardcurves is demonstrated in Example 15. The standard curve may compriseGDF-8 of known concentrations diluted in a buffer. In certainembodiments the buffer is serum, such as, e.g., human serum, mouseserum, primate serum, bovine serum, or ovine serum. The serum isoptionally depleted of endogenous GDF-8 prior to the addition of knownconcentrations of GDF-8. The serum may be obtained from Belgian Bluecattle, which is naturally devoid of GDF-8.

In one embodiment, a method for quantifying free GDF-8 in a biologicalsample comprises combining a GDF-8 capture antibody or antigen bindingprotein and a biological sample under conditions which allow GDF-8, whenpresent in the biological sample, to bind to the one or more captureantibodies forming a capture antibody or antigen binding protein-GDF-8complex; adding a labeled GDF-8 detection antibody or antigen bindingprotein to the solid support from step (b); (d) detecting complexesformed between the capture antibody-GDF-8 complex and the detectionantibody by detecting a signal generated by the label on the GDF-8detection antibody; and (e) quantifying the level of GDF-8 in thebiological sample by comparing the signal generated by complexescontaining the labeled GDF-8 detection antibody to a standard curvegenerated by determining the corresponding signal intensities for knownamounts of GDF-8.

Methods to quantify total GDF-8 are similar, except that the biologicalsample is diluted in acidic buffer.

Methods of Treating, Ameliorating, Preventing, and Inhibiting theProgress of GDF8-Associated Disorders

The involvement of GDF8 in development and/or regulation ofGDF8-associated disorders, e.g., skeletal muscle, bone, glucosehomeostasis, etc., and the discovery of the novel specific GDF8antagonists of the invention enable methods for treating, amelioratingor preventing GDF8-associated disorders, e.g., muscle disorders,neuromuscular disorders, bone-degenerative disorders, metabolic orinduced bone disorders, glucose metabolism disorders, adipose disorders,and insulin-related disorders. In addition, the antagonists allow fordiagnosing, prognosing and monitoring the progress of such disorders bymeasuring the level of GDF8 in a biological sample. In particular,antagonists epitope(s) specific to GDF8 (e.g., peptide mimetics thereto,inhibitory polynucleotides thereto, antibodies thereto, small molecules,etc.) of the invention can be used to treat an individual with a GDF8associated disorder, or in a method of distinguishing whether a patientis suffering from a GDF8-associated disorder.

The antagonists of the present invention are useful to prevent,diagnose, or treat various medical GDF8 associated disorders in humansor animals. The antagonists can be used to inhibit, reduce and/orneutralize one or more activities associated with GDF8. Most preferably,the antagonists inhibit or reduce one or more of the activities of GDF8relative to GDF8 that is not in the presence of an antagonist of theinvention. In certain embodiments, an antagonist of the inventioninhibits the activity of GDF8 by at least 50%, preferably at least 60,62, 64, 66, 68, 70, 72, 72, 76, 78, 80, 82, 84, 86, or 88%, morepreferably at least 90, 91, 92, 93, or 94%, and even more preferably atleast 95% to 100% relative to a mature GDF8 protein that is not bound byone or more anti-GDF8 antibodies. Inhibition or neutralization of GDF8activity can be measured, e.g., in pGL3(CAGA)12 reporter gene assays(RGA) as described in Thies et al., supra, and in ActRIIB receptorassays as illustrated in the Examples.

The medical disorders diagnosed, prognosed, monitored, treated,ameliorated or prevented by the presently disclosed antagonists are GDF8associated disorders, e.g., muscle or neuromuscular disorders including,e.g., muscular dystrophy (MD; including Duchenne's muscular dystrophy),amyotrophic lateral sclerosis (ALS), muscle atrophy, organ atrophy,frailty, carpal tunnel syndrome, congestive obstructive pulmonarydisease, sarcopenia, cachexia, and muscle wasting syndromes (e.g.,caused by other diseases and conditions). In addition, other medicaldisorders that may be diagnosed, prognosed, monitored, treated,ameliorated or prevented by the GDF8 antibodies are adipose tissuedisorders such as obesity, type 2 diabetes, impaired glucose tolerance,metabolic syndromes (e.g., syndrome X), insulin resistance, induced bytrauma (such as burns or nitrogen imbalance), or bone-degenerativediseases (e.g., osteoarthritis and osteoporosis). In preferred, butnonlimiting, embodiments of the invention, the medical disorders thatare diagnosed, prognosed, monitored, treated, ameliorated or preventedby the presently disclosed antagonists are muscular or neuromusculardisorders. In a more preferred, but nonlimiting, embodiment of theinvention, the muscular or neuromuscular disorder that is diagnosed,prognosed, monitored, treated, ameliorated or prevented by the presentlydisclosed antagonists is either MD or ALS.

Other medical disorders that may be diagnosed, treated, ameliorated orprevented by the presently disclosed antagonists are those associatedwith a loss of bone, which include osteoporosis, especially in theelderly and/or postmenopausal women, glucocorticoid-inducedosteoporosis, osteopenia, osteoarthritis, and osteoporosis-relatedfractures. Other target metabolic bone diseases and disorders includelow bone mass due to chronic glucocorticoid therapy, premature gonadalfailure, androgen suppression, vitamin D deficiency, secondaryhyperparathyroidism, nutritional deficiencies, and anorexia nervosa. Theantagonists of the invention are preferably used to prevent, diagnose,ameliorate or treat such medical disorders in mammals, particularly inhumans, e.g., women who will be or are pregnant.

The antagonists of the present invention are administered intherapeutically effective amounts. Generally, a therapeuticallyeffective amount may vary with the subject's age, condition, and sex, aswell as the severity of the medical condition in the subject. The dosagemay be determined by a physician and adjusted, as necessary, to suitobserved effects of the treatment. Toxicity and therapeutic efficacy ofsuch compounds can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of a population) and the ED50 (the dosetherapeutically effective in 50% of a population). The dose ratiobetween toxic and therapeutic effects, i.e., the LD50/ED50, is thetherapeutic index, and antagonists exhibiting large therapeutic indicesare preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that includes the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the form of dosage andthe route of administration. For any antagonist used in the presentinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(e.g., the concentration of the test antagonist which achieves ahalf-maximal inhibition of symptoms or biological activity) asdetermined in cell culture. Levels in plasma may be measured, forexample, by high performance liquid chromatography. The effects of anyparticular dosage can be monitored by a suitable bioassay. Examples ofsuitable bioassays include, but are not limited to, DNA replicationassays, transcription-based assays, GDF8 protein/receptor bindingassays, creatine kinase assays, assays based on the differentiation ofpreadipocytes, assays based on glucose uptake in adipocytes, andimmunological assays.

Generally, the compositions are administered so that antagonists ortheir binding fragments are given at a dose from 1 μg/kg to 150 mg/kg, 1μg/kg to 100 mg/kg, 1 μg/kg to 50 mg/kg, 1 μg/kg to 20 mg/kg, 1 μg/kg to10 mg/kg, 1 μg/kg to 1 mg/kg, 10 μg/kg to 1 mg/kg, 10 μg/kg to 100μg/kg, 100 μg to 1 mg/kg, and 500 μg/kg to 1 mg/kg. Preferably, theantagonists are given as a bolus dose to maximize the circulating levelsof antagonists for the greatest length of time after the dose.Continuous infusion may also be used before, after or in place of thebolus dose.

Methods of Identifying Therapeutic Agents for GDF8-Associated Disorders

Yet another aspect of the invention provides a method of identifyingtherapeutic agents useful in treatment of, e.g., muscle, glucosemetabolism, adipose, and bone disorders. Appropriate screening assays,e.g., ELISA-based assays, are known in the art. In such a screeningassay, a first binding mixture is formed by combining an antagonist,particularly a peptide mimetic of an epitope specific to GDF8 or anantibody or antigen binding protein of the invention and its ligand,GDF8, and the amount of binding between the ligand and the antibody inthe first binding mixture (M0) is measured. A second binding mixture isalso formed by combining the antagonist, the ligand, and a compound oragent to be screened, and the amount of binding between the ligand andthe antibody in the second binding mixture (M1) is measured. The amountsof binding in the first and second binding mixtures are then compared,for example, by calculating the M1/M0 ratio. The compound or agent isconsidered to be capable of specifically interacting with GDF8 if adecrease in binding in the second binding mixture as compared to thefirst binding mixture is observed (i.e., M1/M0<1). The formulation andoptimization of binding mixtures is within the level of skill in theart; such binding mixtures may also contain buffers and salts necessaryto enhance or to optimize binding, and additional control assays may beincluded in the screening assay of the invention.

Compounds found to reduce the antagonist-ligand binding by at leastabout 10% (i.e., M1/M0<0.9), preferably greater than about 30%, may thusbe identified and then, if desired, secondarily screened for thecapacity to inhibit GDF8 activity in other assays such as the ActRIIBbinding assay, or other cell-based and in vivo assays as described inthe Examples or well known in the art.

Small Molecules

Inhibiting GDF8 activity in an organism (or subject) afflicted with (orat risk for) a GDF8-associated disorder, or in a cell from such anorganism involved in such disorders, may also be achieved through theuse of antagonist small molecules (usually organic small molecules) thatantagonize, i.e., inhibit the activity of, GDF8. Novel antagonisticsmall molecules may be identified by the screening methods describedabove and may be used in the treatment methods of the present inventiondescribed herein.

Conversely, increasing GDF8 activity in an organism (or subject)afflicted with (or at risk for) a disorder related to decreased GDF8expression and/or activity or a disorder related to decreased GDF8levels may also be achieved through the use of small molecules (usuallyorganic small molecules) that agonize, i.e., enhance the activity of,GDF8. Novel agonistic small molecules may be identified by screeningmethods and may be used in the treatment methods of the presentinvention described herein.

Methods of Diagnosing, Prognosing, and Monitoring the Progress ofGDF8-Associated Disorders

In addition to treating e.g., muscle, bone, glucose metabolism, andadipose disorders, the present invention provides methods for diagnosingsuch disorders by detecting the decrease or increase of GDF8 in abiological sample, e.g., serum, plasma, bronchoalveolar lavage fluid,sputum, biopsies (e.g., of muscle tissue) etc. “Diagnostic” or“diagnosing” means identifying the presence or absence of a pathologiccondition. Diagnostic methods involve detecting the presence of GDF8 by,e.g., determining a test amount of GDF8 polypeptide in a biologicalsample from a subject (human or nonhuman mammal), and comparing the testamount with a normal amount or range (e.g., an amount or range from anindividual(s) known not to suffer from such a disorder) for the GDF8polypeptide. While a particular diagnostic method may not provide adefinitive diagnosis of GDF8-associated disorders, it suffices if themethod provides a positive indication that aids in diagnosis.

The present invention also provides methods for prognosingGDF8-associated disorders, e.g., muscle, bone, glucose metabolism, andadipose disorders, by detecting upregulation of GDF8. “Prognostic” or“prognosing” means predicting the probable development and/or severityof a pathologic condition. Prognostic methods involve determining thetest amount of GDF8 in a biological sample from a subject, and comparingthe test amount to a prognostic amount or range (e.g., an amount orrange from individuals with varying severities of, e.g., ALS) for GDF8.Various amounts of the GDF8 in a test sample are consistent with certainprognoses for GDF8-associated disorders. The detection of an amount ofGDF8 at a particular prognostic level provides a prognosis for thesubject.

The present invention also provides methods for monitoring the course ofGDF8-associated muscle, bone, glucose metabolism, and adipose disordersby detecting the upregulation or downregulation of GDF8. Monitoringmethods involve determining the test amounts of GDF8 in biologicalsamples taken from a subject at a first and second time, and comparingthe amounts. A change in amount of GDF8 between the first and secondtime indicates a change in the course of, e.g., severity of,GDF8-associated disorders. A skilled artisan will recognize that inGDF8-associated disorders where an increase in muscle mass is desirable,a decrease in amount of GDF8 protein and/or activity between the firstand second time indicates remission of the disorder, and an increase inamount indicates progression of the disorder. Conversely, forGDF8-associated disorders where a decrease in muscle mass is desirable,a decrease in amount in GDF8 protein and/or activity between the firstand second time indicates progression of the disorder, and an increasein amount indicates remission of the disorder. Such monitoring assaysare also useful for evaluating the efficacy of a particular therapeuticintervention (e.g., disease attenuation and/or reversal) in patientsbeing treated for GDF8-associated disorders.

The antagonists of the present invention may be used for diagnosis,prognosis or monitoring by detecting the presence of GDF8 in vivo or invitro. Such detection methods are well known in the art and includeELISA, radioimmunoassay, immunoblot, Western blot, immunofluorescence,immunoprecipitation, and other comparable techniques. The antagonistsmay further be provided in a diagnostic kit that incorporates one ormore of these techniques to detect GDF8. Such a kit may contain othercomponents, packaging, instructions, or other material to aid thedetection of the protein and use of the kit.

Where the antagonists are intended for diagnostic, prognostic, ormonitoring purposes, it may be desirable to modify them, for example,with a ligand group (such as biotin) or a detectable marker group (suchas a fluorescent group, a radioisotope or an enzyme). If desired, theantagonists (whether polyclonal or monoclonal) may be labeled usingconventional techniques. Suitable labels include fluorophores,chromophores, radioactive atoms, electron-dense reagents, enzymes, andligands having specific binding partners. Enzymes are typically detectedby their activity. For example, horseradish peroxidase can be detectedby its ability to convert tetramethylbenzidine (TMB) to a blue pigment,quantifiable with a spectrophotometer. Other suitable labels may includebiotin and avidin or streptavidin, IgG and protein A, and the numerousreceptor-ligand couples known in the art. Other permutations andpossibilities will be readily apparent to those of ordinary skill in theart, and are considered as equivalents within the scope of the instantinvention.

Pharmaceutical Compositions and Methods of Administration

The present invention provides compositions comprising the presentlydisclosed antagonists of the invention, i.e., polypeptides,polynucleotides, vectors, antibodies, antibody or antigen bindingprotein fragments, and small molecules. Such compositions may besuitable for pharmaceutical use and administration to patients. Thecompositions typically comprise one or more molecules of the presentinvention, preferably an antibody or antigen binding protein, and apharmaceutically acceptable excipient. The antagonists of the presentinvention can be used in vitro, ex vivo, or incorporated into apharmaceutical composition when combined with a pharmaceuticallyacceptable carrier. As used herein, the phrase “pharmaceuticallyacceptable excipient” includes any and all solvents, solutions, buffers,dispersion medias, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, that arecompatible with pharmaceutical administration. Such a composition maycontain, in addition to the antagonists of the invention and carrier,various diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials well known in the art. The term “pharmaceuticallyacceptable” means a nontoxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The characteristics of the carrier will depend on the route ofadministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. The compositions may alsocontain other active compounds providing supplemental, additional, orenhanced therapeutic functions. The pharmaceutical compositions may alsobe included in a container, pack, or dispenser together withinstructions for administration.

The pharmaceutical composition of the invention may be in the form of aliposome in which an antagonist of the invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids that exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers while inaqueous solution. Suitable lipids for liposomal formulation include,without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids, and the like.Preparation of such liposomal formulations is within the level of skillin the art.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, e.g.,amelioration of symptoms of, healing of, or increase in rate of healingof such conditions. When applied to an individual active ingredient,administered alone, the term refers to that ingredient alone. Whenapplied to a combination, the term refers to combined amounts of theactive ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of, e.g., an antagonist specific forGDF8 is administered to a subject, e.g., mammal (e.g., a human). Anantagonist of the invention may be administered in accordance with themethod of the invention either alone or in combination with othertherapies such as anti-inflammatory agents. When coadministered with oneor more agents, an antagonist of the invention may be administeredeither simultaneously with the second agent, or sequentially. Ifadministered sequentially, the attending physician will decide on theappropriate sequence of administering an antagonist of the invention incombination with other agents.

In one embodiment, the antagonists of the invention, e.g.,pharmaceutical compositions thereof, are administered in combinationtherapy, i.e., combined with other agents, e.g., therapeutic agents,that are useful for treating pathological conditions or disorders, suchas muscle disorders, neuromuscular disorders, bone degenerativedisorders, metabolic or induced bone disorders, adipose disorders,glucose metabolism disorders or insulin-related disorders, e.g., as wellas allergic and inflammatory disorders. The term “in combination” inthis context means that the agents are given substantiallycontemporaneously, either simultaneously or sequentially. If givensequentially, at the onset of administration of the second compound, thefirst of the two compounds is preferably still detectable at effectiveconcentrations at the site of treatment or in the subject.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods toaccomplish the administration are known to those of ordinary skill inthe art. It may also be possible to obtain compositions which may betopically or orally administered, or which may be capable oftransmission across mucous membranes. Administration of an antagonist ofthe invention used in a pharmaceutical composition to practice themethod of the present invention can be carried out in a variety ofconventional ways, such as oral ingestion, inhalation, cutaneous,subcutaneous, or intravenous injection.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Suchpreparations may be enclosed in ampoules, disposable syringes ormultiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable pharmaceuticallyacceptable carriers include physiological saline, bacteriostatic water,Cremophor™ EL (BASF, Parsippany, N.J.) or phosphate buffered saline(PBS). In all cases, the composition must be sterile and should be fluidto the extent that easy syringability exists. A pharmaceuticallyacceptable carrier must be stable under the conditions of manufactureand storage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, and sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

When a therapeutically effective amount of an antibody or antigenbinding protein of the invention is administered by, e.g., intravenous,cutaneous or subcutaneous injection, the binding agent will be in theform of a pyrogen-free, parenterally acceptable aqueous solution. Thepreparation of such parenterally acceptable protein solutions, havingdue regard to pH, isotonicity, stability, and the like, is within theskill in the art. A preferred pharmaceutical composition forintravenous, cutaneous, or subcutaneous injection should contain, inaddition to binding agents, an isotonic vehicle such as sodium chlorideinjection, Ringer's injection, dextrose injection, dextrose and sodiumchloride injection, lactated Ringer's injection, or other vehicle asknown in the art. The pharmaceutical composition(s) of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additive known to those of skill in the art.

The amount of an antagonist of the invention in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments undergone by the patient. Ultimately, the attending physicianwill decide the amount of antagonist with which to treat each individualpatient. Initially, an attending physician administers low doses of theantagonist and observes the patient's response. Larger doses ofantagonist may be administered until the optimal therapeutic effect isobtained for the patient, and at that point the dosage is generally notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.1 mg to 50 μg antagonist per kg body weight.

The duration of therapy using the pharmaceutical composition of thepresent invention will vary, depending on the severity of the diseasebeing treated and the condition and potential idiosyncratic response ofeach individual patient. It is contemplated that the duration of eachapplication of antagonist will be via, e.g., the subcutaneous route and,e.g., in the range of once a week. Ultimately the attending physicianwill decide on the appropriate duration of therapy using thepharmaceutical composition of the present invention.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, theantagonist (e.g., antibody or antigen binding protein, small molecule,etc.) of the invention can be incorporated with excipients and used inthe form of tablets or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, and the like can contain anyof the following ingredients, or compounds of a similar nature; a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose; a disintegrating agent such asalginic acid, Primogel™, or corn starch; a lubricant such as magnesiumstearate or Sterotes™; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring.

When a therapeutically effective amount of a pharmaceutical compositionof the invention, e.g., an antagonist specific for GDF8, is administeredorally, the binding agent will be in the form of a tablet, capsule,powder, solution or elixir. When administered in tablet form, thepharmaceutical composition of the invention may additionally contain asolid carrier such as a gelatin or an adjuvant. The tablet, capsule, andpowder contain from about 5 to 95% binding agent, and preferably fromabout 25 to 90% binding agent. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added (after taking into account the allergies ofthe individual patient and/or a large population of individuals to suchliquid carriers). The liquid form of the pharmaceutical composition mayfurther contain physiological saline solution, dextrose or othersaccharide solution, or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol. When administered in liquid form, thepharmaceutical composition contains from about 0.5 to 90% by weight ofthe binding agent, and preferably from about 1 to 50% the binding agent.

For administration by inhalation, an antagonist of the invention isdelivered in the form of an aerosol spray from a pressured container ordispenser, which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer. Accordingly, the compounds describedherein can be administered by inhalation to pulmonary tissue. The term“pulmonary tissue” as used herein refers to any tissue of therespiratory tract and includes both the upper and lower respiratorytract, except where otherwise indicated. A specific GDF8 antagonist canbe administered in combination with one or more of the existingmodalities for treating pulmonary diseases.

In one example of administration, the compound is formulated for anebulizer. In one embodiment, the compound can be stored in alyophilized form (e.g., at room temperature) and reconstituted insolution prior to inhalation.

It is also possible to formulate the compound for inhalation using amedical device, e.g., an inhaler (see, e.g., U.S. Pat. Nos. 6,102,035 (apowder inhaler) and 6,012,454 (a dry powder inhaler)). The inhaler caninclude separate compartments for the active compound at a pH suitablefor storage and another compartment for a neutralizing buffer, and amechanism for combining the compound with a neutralizing bufferimmediately prior to atomization. In one embodiment, the inhaler is ametered dose inhaler.

Although not necessary, delivery enhancers such as surfactants can beused to further enhance pulmonary delivery. A “surfactant” as usedherein refers to a compound having hydrophilic and lipophilic moietiesthat promote absorption of a drug by interacting with an interfacebetween two immiscible phases. Surfactants are useful with dry particlesfor several reasons, e.g., reduction of particle agglomeration,reduction of macrophage phagocytosis, etc. When coupled with lungsurfactant, a more efficient absorption of the compound can be achievedbecause surfactants, such as DPPC, will greatly facilitate diffusion ofthe compound. Surfactants are well known in the art and include, but arenot limited to, phosphoglycerides, e.g., phosphatidylcholines,L-alpha-phosphatidylcholine dipalmitoyl (DPPC) and diphosphatidylglycerol (DPPG); hexadecanol; fatty acids; polyethylene glycol (PEG);polyoxyethylene-9-; auryl ether; palmitic acid; oleic acid; sorbitantrioleate (Span 85); glycocholate; surfactin; poloxomer; sorbitan fattyacid ester; sorbitan trioleate; tyloxapol; and phospholipids.

Systemic administration can also be by transmucosal or transdermalmeans. For example, in the case of antibodies that comprise the Fcportion, compositions may be capable of transmission across mucousmembranes (e.g., intestine, mouth, or lungs) via the FcRnreceptor-mediated pathway (e.g., U.S. Pat. No. 6,030,613). In general,transmucosal administration can be accomplished, for example, throughthe use of lozenges, nasal sprays, inhalers, or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, patches or creams as generally known in theart. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, detergents, bile salts, and fusidic acid derivatives.

Pharmaceutical compositions may also consist of compositions suitablefor gene therapy, i.e., compositions comprised of the polynucleotidesdisclosed herein. In the case of gene therapy, the pharmaceuticallyacceptable carrier may include, e.g., lipids, collagen spheres, cationicemulsion systems, water, saline buffers, viral vectors, chylomicronremnants, polymer nanoparticles (e.g., gelatin-DNA or chitosan-DNA),gold particles, polymer complexes, lipoplexes, polyplexes, etc. (see,e.g., Gardlik et al. (2005) Med. Sci. Monit. 11(4):RA110-21).

Stabilization and Retention

In one embodiment, a specific GDF8 antagonist is physically associatedwith a moiety that improves its stabilization and/or retention incirculation, e.g., in blood, serum, lymph, bronchopulmonary orbronchoalveolar lavage, or other tissues, e.g., by at least 1.5, 2, 5,10, or 50 fold.

The presently disclosed antagonists of the invention may be preparedwith carriers that will protect against rapid elimination from the body,such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. Liposomal suspensions containing the presentlydisclosed antagonists can also be used as pharmaceutically acceptablecarriers. These can be prepared according to methods known to thoseskilled in the art.

For example, a specific GDF8 antagonist can be associated with apolymer, e.g., a substantially nonantigenic polymer, such aspolyalkylene oxides or polyethylene oxides. Suitable polymers will varysubstantially by weight. Polymers having molecular number averageweights ranging from about 200 to about 35,000 (or about 1,000 to about15,000, or about 2,000 to about 12,500) can be used.

For example, a specific GDF8 antagonist can be conjugated to awater-soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g.,polyvinylalcohol and polyvinylpyrrolidone. A nonlimiting list of suchpolymers include polyalkylene oxide homopolymers such as polyethyleneglycol (PEG) or polypropylene glycols, polyoxyethylenated polyols,copolymers thereof and block copolymers thereof, provided that the watersolubility of the block copolymers is maintained. Additional usefulpolymers include polyoxyalkylenes such as polyoxyethylene,polyoxypropylene, and block copolymers of polyoxyethylene andpolyoxypropylene (Pluronics); polymethacrylates; carbomers; branched orunbranched polysaccharides, which comprise the saccharide monomersD-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose,D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid(e.g., polymannuronic acid, or alginic acid), D-glucosamine,D-galactosamine, D-glucose and neuraminic acid includinghomopolysaccharides and heteropolysaccharides such as lactose,amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate,dextran, dextrins, glycogen, or the polysaccharide subunit of acidmucopolysaccharides, e.g., hyaluronic acid; polymers of sugar alcoholssuch as polysorbitol and polymannitol; heparin; etc.

Other compounds can also be attached to the same polymer, e.g., acytotoxin, a label, or another targeting agent, e.g., another GDF8antagonist or an unrelated ligand. Mono-activated, alkoxy-terminatedpolyalkylene oxides (PAOs), e.g., monomethoxy-terminated polyethyleneglycols (mPEGs), C1-4 alkyl-terminated polymers, and bis-activatedpolyethylene oxides (glycols) can be used for cross-linking (see, e.g.,U.S. Pat. No. 5,951,974).

In one embodiment, the polymer prior to cross-linking to the ligand neednot be, but preferably is, water-soluble. Generally, aftercross-linking, the product is water-soluble, e.g., exhibits a watersolubility of at least about 0.01 mg/ml, and more preferably at leastabout 0.1 mg/ml, and still more preferably at least about 1 mg/ml. Inaddition, the polymer should not be highly immunogenic in the conjugateform, nor should it possess viscosity that is incompatible withintravenous infusion, aerosolization, or injection, if the conjugate isintended to be administered by such routes.

In one embodiment, the polymer contains only a single group that isreactive. This helps to avoid cross-linking of ligand molecules to oneanother. However, it is within the scope herein to maximize reactionconditions to reduce cross-linking between ligand molecules, or topurify the reaction products through gel filtration or ion exchangechromatography to recover substantially homogenous derivatives. In otherembodiments, the polymer contains two or more reactive groups for thepurpose of linking multiple ligands to the polymer backbone. Again, gelfiltration or ion exchange chromatography can be used to recover thedesired derivative in substantially homogeneous form.

The molecular weight of the polymer can range up to about 500,000 D, andpreferably is at least about 20,000 D, or at least about 30,000 D, or atleast about 40,000 D. The molecular weight chosen can depend upon theeffective size of the conjugate to be achieved, the nature (e.g.,structure, such as linear or branched) of the polymer, and the degree ofderivatization.

A covalent bond can be used to attach a specific GDF8 antagonist to apolymer, for example, cross-linking to the N-terminal amino group of theligand and epsilon amino groups found on lysine residues of the ligand,as well as other amino, imino, carboxyl, sulfhydryl, hydroxyl or otherhydrophilic groups. The polymer may be covalently bonded directly to theGDF8 antagonist without the use of a multifunctional (ordinarilybifunctional) cross-linking agent. Covalent binding to amino groups isaccomplished by known chemistries based upon cyanuric chloride, carbonyldiimidazole, and aldehyde-reactive groups (PEG alkoxide plus diethylacetyl of bromoacetaldehyde, PEG plus DMSO and acetic anhydride, or PEGchloride plus the phenoxide of 4-hydroxybenzaldehyde, activatedsuccinimidyl esters, activated dithiocarbonate PEG,2,4,5-trichlorophenylcloroformate or P-nitrophenylchloroformateactivated PEG). Carboxyl groups can be derivatized by coupling PEG-amineusing carbodiimide. Sulfhydryl groups can be derivatized by coupling tomaleimido-substituted PEG (e.g., alkoxy-PEG amine plus sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (see WO 97/10847) orPEG-maleimide). Alternatively, free amino groups on the ligand (e.g.,epsilon amino groups on lysine residues) can be thiolated with2-imino-thiolane (Traut's reagent) and then coupled tomaleimide-containing derivatives of PEG, e.g., as described in Pedley etal. (1994) Br. J. Cancer 70:1126-30.

Functionalized PEG polymers that can be attached to a GDF8 antagonistare available, e.g., from Shearwater Polymers, Inc. (Huntsville, Ala.).Such commercially available PEG derivatives include, e.g., amino-PEG,PEG amino acid esters, PEG-hydrazide, PEG-thiol, PEG-succinate,carboxymethylated PEG, PEG-propionic acid, PEG amino acids, PEGsuccinimidyl succinate, PEG succinimidyl propionate, succinimidyl esterof carboxymethylated PEG, succinimidyl carbonate of PEG, succinimidylesters of amino acid PEGs, PEG-oxycarbonylimidazole, PEG-nitrophenylcarbonate, PEG tresylate, PEG-glycidyl ether, PEG-aldehyde, PEGvinylsulfone, PEG-maleimide, PEG-orthopyridyl-disulfide,heterofunctional PEGs, PEG vinyl derivatives, PEG silanes, and PEGphospholides. The reaction conditions for coupling these PEG derivativesmay vary depending on the specific GDF8 antagonist, the desired degreeof PEGylation, and the PEG derivative utilized. Some factors involved inthe choice of PEG derivatives include: the desired point of attachment(such as lysine or cysteine R-groups), hydrolytic stability andreactivity of the derivatives, stability, toxicity and antigenicity ofthe linkage, suitability for analysis, etc. Specific instructions forthe use of any particular derivative are available from themanufacturer.

The conjugates of a GDF8 antagonist and a polymer can be separated fromthe unreacted starting materials, e.g., by gel filtration or ionexchange chromatography, or other forms of chromatography, e.g., HPLC.Heterologous species of the conjugates are purified from one another inthe same fashion. Resolution of different species (e.g., containing oneor two PEG residues) is also possible due to the difference in the ionicproperties of the unreacted amino acids (see, e.g., WO 96/34015).

The polynucleotides and proteins of the present invention are expectedto exhibit one or more of the uses or biological activities (includingthose associated with assays cited below) identified herein. Uses oractivities described for proteins of the present invention may beprovided by administration or use of such proteins, or by administrationor use of polynucleotides encoding such proteins (such as, e.g., in genetherapies or vectors suitable for introduction of DNA).

It may be advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated, each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound, the particular therapeuticeffect to be achieved, and the limitations inherent in the art offormulating such an active compound for the treatment of individuals.

Another aspect of the present invention accordingly relates to kits forcarrying out the administration of the GDF8 antagonists of theinvention, e.g., with or without other therapeutic compounds, or forusing the GDF8 antagonists as a research or therapeutic tool todetermine the presence and/or level of GDF8 in a biological sample, suchas an ELISA kit. In one embodiment, the kit comprises one or moreanti-GDF8 antagonists formulated in a pharmaceutical carrier, and atleast one agent, e.g., a therapeutic agent, formulated as appropriate,in one or more separate pharmaceutical preparations.

The Examples which follow are set forth to aid in the understanding ofthe invention but are not intended to, and should not be construed to,limit the scope of the invention in any way. The Examples do not includedetailed descriptions of conventional methods, such as hybridomaformation, ELISA, proliferation assays, flow cytometric analysis andrecombinant DNA techniques. Such methods are well known to those ofordinary skill in the art.

The entire contents of all references, patents, published patentapplications, and other patent documents cited throughout thisapplication are herein incorporated by reference.

The present invention is further illustrated and supported by thefollowing examples. However, these examples should in no way beconsidered to further limit the scope of the invention. To the contrary,one having ordinary skill in the art would readily understand that thereare other embodiments, modifications, and equivalents of the presentinvention without departing from the spirit of the present inventionand/or the scope of the appended claims.

EXAMPLES Example 1 Creation of Hybridoma Cells and Isolation of RK22Anti-GDF8 Antibody

GDF8 (myostatin) knockout mice (McPherron et al. (1997) Proc. Natl.Acad. Sci. U.S.A. 94:12457-61) were immunized by subcutaneous injectionwith Freund's complete adjuvant and 20 μg recombinant GDF8 dimer thatwas purified from CHO cell conditioned media as described in Lee andMcPherron (1999) Curr. Opin. Genet. Dev. 9:604-07. Several boosterinjections of the same amount of GDF8 and Freund's incomplete adjuvantwere given at 2-week intervals. A final intravenous injection in thetail vein of 2 μg PBS was given prior to isolation of splenocytes frommice demonstrating the high titers of anti-GDF8 antibodies. Isolatedsplenocytes were fused with mouse myeloma cells (ATCC Accession No.P3×63.Ag8.653). After 10-14 days the supernatants from hybridomas wereharvested and tested by ELISA for anti-GDF8 antibody levels, see Example2. To ensure monoclonality, hybridomas chosen for further studies werecloned by repeated limiting dilution.

Supernatants from anti-GDF8-expressing hybridomas and/or antibodiespurified from the supernatants using standard affinity chromatographymethods well known in the art, were tested for specificity in assaysdescribed in Example 2. Of thirteen clones initially tested for bindingto GDF8, RK22 was among those selected for further analysis.

Example 2 RK22 Antibody Specifically Interact with GDF8 Example 2.1 RK22has a Higher Affinity for GDF8 than for BMP11 in ELISA Assays

Standard ELISA techniques using either GDF8 or BMP11 were used todetermine the specificity of RK22 binding to GDF8, i.e., to determinewhether the antibodies demonstrated a higher affinity for GDF8 than forBMP11. Recombinant human GDF8 (mature GDF8 and GDF8 propeptide) andBMP11 protein were purified and characterized as previously disclosed inU.S. Patent Published Application No. 2004/0142382. The GDF8 latentcomplex and the BMP11 latent complex were each individually biotinylatedat a ratio of 20 moles of EZ-link Sulfo-NHS-Biotin (Pierce, Rockford,Ill., Cat. No. 21217) to 1 mole of complex for 2 hrs on ice. Thereaction was terminated by decreasing the pH using 0.5% TFA, andbiotinylated complex was subjected to chromatography on a C4 Jupiter250×4.6 mm column (Phenomenex, Torrance, Calif.) to separate matureprotein (e.g., mature GDF8 from GDF8 propeptide or mature BMP11 fromBMP11 propeptide). Fractions of mature biotinylated GDF8 or maturebiotinylated BMP11 eluted with a TFA/CH3CN gradient were pooled,concentrated, and quantified by MicroBCA™ protein assay reagent kit(Pierce, Rockford, Ill., Cat. No. 23235).

A 96-well microtiter plate (precoated overnight at 4° C. with 5 μg/mlstreptavidin in PBS) was coated with 0.5 μg/ml biotinylated GDF8 orbiotinylated BMP11 for 1 hr at room temperature. Excess GDF8 or BMP11was removed by washing with PBS containing 0.1% (v/v) Tween 20 (PBSTbuffer). The plates were blocked for 1 hr at room temperature inSuperBlock™ solution (Pierce) and then rinsed with PBS. GDF8 or BMP11coated plates were incubated at room temperature for 1 hr with 100 μl ofpre-blocked supernatant collected from RK22 hybridomas, or with purifiedRK22 antibody at various concentrations. To control for non-specificbinding the RK35 antibody (see U.S. Patent Application No. 60/709,704,hereby incorporated by reference in its entirety) that has been shown tobind and inhibit both GDF8 and BMP11 was used along with an irrelevantantibody (Irr. Ab) that has demonstrated no binding to either GDF8 orBMP11, and control media were also individually tested. Unbound antibodywas removed by 3 washes of PBST followed by 3 washes with PBS. Fifty μlof a 1:5000 dilution of goat anti-mouse IgG HRP conjugate was added toeach well. The plates were incubated at room temperature for 1 hr. Eachplate was washed three times with PBST, and subsequently, 3 times withPBS and developed for a color reaction by the addition of the TMB(tetramethylbenzidine) reagent. The color reaction was stopped by theaddition of 100 μl of 0.18 M H2SO4. The signal generated was measured byreading the optical density at 450 nm of each well using a microtiterplate reader.

As shown in FIGS. 1A and 1B, the supernatants from RK22 hybridomas hadgreater binding to GDF8 than BMP11 compared to supernatant isolated fromthe control antibody, RK35. The uniformity of higher affinity for GDF8by RK22 was confirmed by demonstrating that the higher affinity for GDF8of these antibodies was not dose dependent. As can be seen in FIGS. 1Aand B, at every concentration tested RK22 antibodies demonstratedgreater binding to GDF8 than to BMP11. RK22 showed very little bindingto BMP11 while in contrast, the RK35 control antibody bound to both GDF8and BMP11.

Example 2.2 Binding Affinities of RK22 for GDF8

Molecular kinetic interactions of RK22 antibody with GDF-8 werequantitatively analyzed using BIAcore plasmon resonance technology, andapparent kinetic rate constants were derived. In these studies wemeasured the binding of soluble antibody to solid phase bound GDF-8. Thesurface orientation of the immobilized GDF-8 onto the biosensor surfacewas controlled using biotinylated GDF-8 (bio-GDF-8), the bio GDF-8 wasimmobilized onto streptavidin biosensor chips then, variousconcentrations of antibody were applied in triplicates and the bindingwas measured as function of time. From these measurements the apparentdissociation (K_(d)) and association (K_(a)) rate constants were derivedand used to calculate a binding affinity constant (K_(d)) for theinteraction. The active concentration of RK22, defined as the fractionof antibody that are biologically functional, was determined bymeasuring the fraction of antibody able to bind to the bio GDF-8immobilized on the chip using the BIAcore under partial mass transportlimitations by coating a high surface density and injecting the antibodyat different flow rates. The association and dissociation rate for eachconcentration of antibody were calculated simultaneously using globalfit with the biaevaluation software version 3.0.2.

The BIAcore 2000 system, Sensor Chip SA (BR-1000-32), HBS/EP buffer(0.01 M HEPES pH.7.4, 0.15 M NaCl, 3.0 mM EDTA and 0.005% polysorbate 20(v/v), N-hydroxysuccinimide (NHS-EP) were obtained from BIAcore AB,Uppsala, Sweden. The human bio-GDF8 (Lot 25251-15) was purified. 0.1%TFA (v/v) (Sigma) was made in water. Experimental data from kineticdeterminations of the antibody-antigen interaction was analyzed usingthe BIAevaluation software version 3.0.2.

To prepare the bio-GDF8 surface, a continuous flow of HBS/EP buffer wasmaintained over the sensor surface. The streptavidin on the sensorsurface was conditioned with 3 injections (1 minute each) of a solutioncontaining 1 M NaCl and 25 mM NaOH. For high-density coating (>2000RU)bio-GDF8 was diluted to 1 ug/ml in HBS/EP buffer and immobilized on thestreptavidin chip by flowing over it. For low-density coating (20-60 RU)the GDF-8 was further diluted to 0.1 ug/ml and the volume of injectedbio-GDF-8 varied according to the density required. The streptavidinsurface on flow cell one was used as reference surface. As control thefirst flow cell was used as reference surface to correct for bulkrefractive index, matrix effects and non-specific binding, the second,third and four flow cells were coated with the capturing molecule.

The fraction of RK22 antibody able to bind to the bio GDF-8 immobilizedon the chip was analyzed using the BIAcore under partial mass transportlimitations. In this experiments anti-GDF-8 antibody at 200 nM and 100nM (concentrations measured based on OD 280) were injected at flow ratesof 2, 10, 30, 50 and 100 ul/min. Mass transport limitations could bedetected by visual inspection of the sensor grams, since the slopesincreased with increasing flow rates. Biosensor surfaces wereregenerated using 5 ul of 0.1% TFA.

Both RK22 anti-GDF-8 antibody were diluted in HBS-EP buffer (BiacoreAB), aliquots were injected over the immobilized bio-GDF-8 at a flowrate of 30 ul/min, following injection for three minutes, dissociationwas monitored in BIAcore buffer for ten minutes at the same flow rate.The concentrations of antibody injected were 300, 150, 75, 37.5, 18.7,9.3, 4.6, 2.3 and 0 nM; each injection was done in triplicate. Blank andbuffer effects were subtracted for each sensorgram using doublereferencing. Biosensor surfaces were regenerated using 5 ul of 0.1% TFA,before the injection of the next sample HBS-EP alone flowed through eachcell. The response was measured in resonance units (RU) representing themass of bound of RK22.

The kinetic data was analyzed using BIAevaluation software 3.0.2.Assuming both a bivalent analyte (A) binding to monovalent ligand (B).A+B=AB K_(a)1*K_(d)1; AB+B=AB2 K_(a)2*K_(d)2; and a monovalent analyte(A) binding to monovalent ligand (B) A+B=AB K_(a)1*K_(d)1. The apparentdissociation (k_(d)) and association (k_(a)) rate constants werecalculated from the appropriate regions of the sensorgrams. The bindingaffinity constant of the interaction between antibody and GDF8 wascalculated from the kinetic rate constants by the following formula:K_(d)=kd/ka. As can be seen in FIG. 2, RK22 demonstrated a K_(d) valueof 7 nM in the average of three experiments.

Example 3 RK22 Inhibit GDF8 Signaling In Vitro and In Vivo Example 3.1Inhibition of the Biological Activity of Purified Recombinant HumanGDF-8 in the Cell Based Reporter Gene Assay Using RK22

To demonstrate the activity of GDF-8 in the in vitro cell based assay, areporter gene assay (RGA) was developed using a reporter vector pGL3(CAGA)12 expressing luciferase under control of TGF-β induced promoter.The CAGA sequence was previously reported to be a TGF-β responsivesequence within the promoter of the TGF-β induced gene PAI-1 (Thies S etal 2001). A reporter vector containing 12 CAGA boxes was made using thebasic luciferase reporter plasmid pGL3 (Promega, Madison, Wis.). TheTATA box and transcription initiation site from the adenovirus majorlater promoter (−35/+10) was inserted between the BgIII and HindIIIsites. Oligonucleotides containing 12 repeats of the CAGA boxesAGCCAGACA were annealed and cloned into the XhoI site. The humanrhabdomyosarcoma cell line A204 (ATCC HTB-82) was transientlytransfected with pGL3 (CAGA)12 using FuGENE 6 transfection reagent(Boehringer Manheim, Germany). Following transfection, cells werecultured on 96 well plates in McCoy's 5A medium supplemented with 2 mMglutamine, 100 U/ml streptomycin, 100 μg/ml penicillin and 10% fetalcalf serum for 16 hrs. Cells were then treated with or without 10 ng/mlGDF-8 in McCoy's 5A media with glutamine, streptomycin, penicillin, and1 mg/ml bovine serum albumin for 6 hrs at 37° C. Luciferase wasquantified in the treated cells using the Luciferase Assay System(Promega). The assay was repeated using 10 ng/ml BMP-11.

To test the inhibitory activity of RK22, GDF-8 was preincubated withRK22 antibody for 1 hour at room temperature. This mixture was thenadded to the transfected cells and were incubated for 6 hrs at 37° C.Luciferase was quantified using the Luciferase Assay System (Promega).The ability of RK22 to block BMP-11 activity was measured using the sameprotocol.

As seen in FIG. 3, induction of pGL3(CAGA)12 reporter activity as LCPSwhen cells were untreated (bkgd) or treated with 10 ng/ml GDF8 in theabsence or presence of RK22. Each of these antibodies reduced at leastone GDF8 activity, i.e., GDF8-mediated luciferase induction, in adose-responsive manner, with an IC50 of 0.4 nM for RK22. The controlantibody RK35 had an IC50 of 0.2 nm, and an irrelevant antibody had anIC50 of >100 nM. Although RK22 inhibited GDF8-mediated signaling, theseantibodies did not significantly inhibit the biological activity ofBMP11; the IC50 for inhibition of BMP11 activity by RK22 was notdetectable, 30 nM, and >100 nM, respectively. These data demonstratethat RK22 inhibit specifically GDF8 signaling in vitro to a similardegree as the nonspecific antibody RK35.

Example 3.2 RK22 Inhibits GDF8 Activity In Vivo

In order to determine whether RK22 antibodies antagonize GDF8 activityin vivo, RK22 was selected as a representative antibody for furthertesting in adult SCID mice. SCID mice suffer from a severe combinedimmune deficiency, and therefore do not generate an immunologicalreaction following injections of antibodies such as RK22. RK22 wasinjected into SCID mice over a period of four weeks. Three dosages ofRK22 were administered: 1 mg/kg/week, 10 mg/kg/week and 40 mg/kg/week.Myo-29 administered at a dose of 10 mg/kg/week was used as a positivecontrol and compared to the different concentrations of RK22administered.

Muscle mass was used as an indicator for GDF8 activity in mice treatedwith RK22. Three different muscle groups, gastroc, tibialis anterior andquad, were removed and muscle weight was determined. As shown in FIG. 4,RK22 significantly increased muscle mass at a dose of 10 mg/kg/week. Incomparison to the positive control, Myo29, the muscle mass increasedapproximately 10% in both 10 mg/kg/week of for both RK22 and Myo29.

Example 4 Characterization of RK22 Binding Sites Example 4.1 Assessmentof RK22 Inhibition of GDF8 Binding to ActRIIB

To determine whether RK22 antibodies are capable of antagonizing GDF8activity by preventing GDF8 from binding its ActRIIB receptor, theantibodies were tested in an ActIIRB binding assay (e.g., aneutralization assay). Purified RK22 antibodies were screened for theability to inhibit the binding of biotinylated GDF8 to ActRIIB fusionprotein immobilized on plastic in a 96-well microtiter plate assay.Recombinant ActRIIB-Fc chimera (R&D Systems, Minneapolis, Minn., Cat.No. 339-RB/CF) was coated onto 96-well flat-bottom assay plates (Costar,N.Y., Cat. No. 3590) at 1 μg/ml in 0.2 M sodium carbonate bufferovernight at 4° C. Plates were then blocked with 1 mg/ml bovine serumalbumin and washed following a standard ELISA protocol. Twenty ng/ml ofbiotinylated GDF8 alone or preincubated for one hour at room temperaturewith various concentrations of RK22 was added to the blocked ELISAplate. To establish clone potency as measured by IC₅₀ values, atitration of antibodies was added. Biotinylated GDF8 preincubated withirrelevant antibody or a control antibody that blocks GDF8 binding toActIIRB were included as controls. After one hour at room temperature,the antibody-blocked protein complexes were washed away, and the amountof GDF8 bound to plate-bound ActIIRB was detected with Europeium-labeledstreptavidin using the DELFIA™ reagent kit (PerkinElmer LifeSciences,Boston, Mass.) in a time-resolved fluorometric (TRF) assay.

The results of the ActRIIB neutralization assay are shown in FIG. 5.Therefore, although it is possible that RK22 antibodies inhibit GDF8signaling by inhibiting the ability of GDF8 to bind to ActRIIB, it ismore likely that another mechanism is involved.

Example 4.2 RK22 Binds to GDF8-Specific Epitopes

Although GDF8 and BMP11 are closely related, RK22 is specific for GDF8.Consequently, it is hypothesized that the epitopes recognized by theseantibodies are also specific to GDF8, and thus, the epitopes specific toGDF8 may be used as antagonists (e.g., as peptide mimetics) tospecifically inhibit GDF8 signaling and/or to screen for or makeGDF8-specific antagonists.

To determine the GDF8 epitopes recognized by antibodies RK22, 48overlapping 13-residue peptides presenting the entire sequence of matureGDF8 set forth as SEQ ID NO:1 were synthesized directly on cellulosepaper using the spot synthesis technique (Molina et al. (1996) PeptideRes. 9:151-55; Frank et al. (1992) Tetrahedron 48:9217-32). The overlapof the peptides was 11 amino acids. In this array, cysteine residueswere replaced with serine in order to reduce the chemical complicationsthat are caused by the presence of cysteines. Cellulose membranesmodified with polyethylene glycol and Fmoc-protected amino acids werepurchased from Abimed (Lagenfeld, Germany). The array was defined on themembrane by coupling a β-alanine spacer, and peptides were synthesizedusing standard DIC (diisopropylcarbodiimide)/HOBt (hydroxybenzotriazole)coupling chemistry as described previously (Molina et al., supra; Franket al., supra).

Activated amino acids were spotted using an Abimed ASP 222 robot.Washing and deprotection steps were done manually and the peptides wereN-terminally acetylated after the final synthesis cycle. Followingpeptide synthesis, the membrane was washed in methanol for 10 min and inblocker (TBST (Tris-buffered saline with 0.1% (v/v) Tween™ 20) and 1%(w/v) casein) for 10 min. The membrane was then incubated with 2.5 μg/mlof RK22 anti-GDF8 antibody in blocker for one hour with gentle shaking.After washing with blocker 3 times for 10 min, the membrane wasincubated with HRP-labeled secondary antibody (0.25 μg/ml in blocker)for 30 min. The membrane was then washed three times for 10 min eachwith blocker and 2 times for 10 min each with TBST. Bound antibody wasvisualized using SUPERSIGNAL™ West reagent (Pierce) and a digital camera(Alphananotech Fluoromager). The dot blots are shown in FIG. 6 and theresults summarized in Table 4. The dot blots demonstrate that RK22 bindsto a GDF8 epitope(s) having and/or consisting essentially of an aminoacid sequence selected from the group consisting of: DFGLDS (SEQ IDNO:4), FEAFGWDWIIAPKRY (SEQ ID NO:6), FVFLQKYPHTLVHQ (SEQ ID NO:8),SSGESEFVF (SEQ ID NO:10), WIIAPKRYKANYSSGESEFVFLQKY (SEQ ID NO:11), andpotentially subsequences thereof. In particular, the epitope(s) for RK22maps to a GDF8 region that putatively interacts with the GDF8 Type Ireceptor (ALK4/ALK5).

TABLE 4 Approximate regions of human GDF8 bound by Mouse MonoclonalAntibodies RK22 Epitope Area N-terminal and Type I receptor recognitionregions Interaction with Amino 1-6; 24-38; 49-63 Acids of SEQ ID NO: 1(approximate)

Example 5 Humanization of RK22 Example 5.1 Antibody Sequencing

The variable heavy (VH) and variable light (VL) genes encoding RK22 werecloned from the hybridoma cells producing the RK22 antibody and then theamino acid sequences determined. These sequences are listed in Table 1as SEQ ID NOs: 14 and 16.

Example 5.2 Germlining RK22 Antibody

Sequence data for the antibodies was used to identify the nearestgermline sequence for the heavy and light chain of RK22, e.g., DP-5 andDP-7 displayed about 65% and 71% identity to RK22 VH, respectively (FIG.7); while DPK 24 displayed about 78% identity to RK22 VL (FIG. 8).Appropriate mutations were made using standard site directed mutagenesistechniques with the appropriate mutagenic primers. Mutation of sequencesand antibodies was confirmed by sequence analysis. The specification ismost thoroughly understood in light of the teachings of the referencescited within the specification, all of which are hereby incorporated byreference in their entirety. The embodiments within the specificationprovide an illustration of embodiments of the invention and should notbe construed to limit the scope of the invention. The skilled artisanrecognizes that many other embodiments are encompassed by the claimedinvention.

Example 6 Sandwich Immunoassay Formats for Quantifying and DetectingGDF8

Each of the antibodies described in the Examples below were biotinylatedusing an EZ Link Sulfo-LC biotinylation kit from Pierce. From previousstudies, it was determined that a 40-fold excess of NHS-biotin wasoptimal for biotinylation of both of these antibodies. 400 ug of eachantibody was biotinylated using a 40 fold molar excess biotin. Thisresulted in an incorporation of approximately 3 to 5 mmoles of biotinper mmole of antibody. After biotinylation, all antibodies were dialyzedinto PBS overnight at 4° C. and total protein concentration determinedby BCA. To determine the molar ratio of biotin incorporation, a solutionof the biotinylated antibody was added to a mixture of2-(4′-Hydroxyazobenzene) benzoic acid (HABA) and avidin. Because of itshigher affinity for avidin, biotin displaced the HABA from itsinteraction with avidin and the absorbance at 500 nm decreasedproportionately. The amount of biotin conjugated to an antibody can bequantitated by measuring the absorbance of the HABA-avidin solutionbefore and after the addition of the biotinylated sample. The change inabsorbance relates to the amount of biotin incorporated into theantibody.

Additionally, the serum used in the studies described below is devoid ofendogenous GDF8. An affinity column for GDF-8 serum depletion wasprepared using 1 mg of MYO-029 monoclonal antibody immobilized ontocyanogen bromide activated Sepharose beads. The column was pre-washedwith 0.1 M acetic acid and neutralized with PBS containing 250 mM NaClpH 7.2 before the addition of human serum. Due to the apparentactivation of latent GDF-8 by heating to 65° C., serum was preheated to65° C. for ten minutes and then passed three times over the 1 mg MYO-029anti-GDF8 affinity column and checked for activity in the free and totalGDF-8 assays. Initially 2.5 ml volumes of serum were passed over thecolumn. After initial testing, larger volumes of serum (13 ml aliquots)were heated and depleted of GDF-8 by multiple passes over the affinitycolumn with intermediate column washes in 0.1 M acetic acid. Thisdepleted serum was used as the matrix for generation of standard curveswith known concentrations of mature GDF-8.

Example 6.1 Antibody Pairing Experiments: Comparison of ImmunoassayFormats: RK35 Capture with RK22 Detector or RK22 Capture with RK35Detector

Each anti-GDF-8 monoclonal antibody was individually coated in 0.1 Msodium borate on a high binding, 96-well plate (Immulon 4 HBX) overnightat 4° C. or for 1 hour at 37° C. at a concentration of 1 μg/ml. Theplate was washed and then blocked for ten minutes with Pierce Superblockreagent. Stock GDF-8 (1.77 mg/ml in 0.1% trifluoroacetic acid-TFA) wasdiluted to 10 μg/ml in 0.1% trifluoroacetic acid (TFA) in siliconizedplastic tubes and further diluted into GDF8 depleted human serum atconcentrations ranging from 12.5 ng/ml to 0.2 ng/ml as calibrators forgeneration of standard curves. The standard curve was run in triplicateusing columns 1-3 of the assay plate. In a separate pre-blocked, 96-wellplate, 30 μl aliquots of test serum was added to each of 6 wells (fortriplicate determinations) containing 120 μl of THST buffer (finalconcentration, 20% serum; total volume, 150 μl). For the total GDF-8assay, 80 μl of this solution was transferred to a 96-well PCR plate andheated to 80° C. for 5 minutes. The heated samples were cooled on icebefore addition to the assay plate.

To the wells used for the standard curve, 65 μl of THST buffer was addedto all wells followed by 10 μl of the calibrator serum, to bring thefinal volume to 100 μl. From the preparation plate, 50 μl of unheated20% serum sample was removed and added to the assay plate for a finalserum concentration of 10% serum in a total volume of 100 μl. From theheated plate, 50 μl of 20% serum was added to a second assay plate.These second assay plates were incubated at room temperature withshaking. After 1.5 hours, the plates were washed and Superblock reagentwas added for 5 minutes. Biotinylated RK22 or biotinylated RK35 wasadded to the wells at 150 ng/ml for 1.5 hours with shaking. The plateswere washed and re-blocked again before the addition of 100 ul ofultrasensitive strepavidin-HRP (1:20,000) dilution for 1 hour at roomtemperature with shaking. Plates were washed and re-blocked before theaddition of TMB substrate for 15 minutes at room temperature withshaking. The reaction was stopped by the addition of 0-5 M H2S04 andread on a Molecular Devices Spectramax plate reader at 450 nm. Acomparison of the immunoassay formats: RK35 capture/RK22 detector orRK22 capture/RK35 detector can be seen in FIGS. 9A and 9B. As shown,either format was capable of detecting between 100 and 10 pg/ml of GDF8in THST assay buffer or in 10% serum.

Example 6.2 Effects of Serum on the GDF-8 Assay

Previous results suggested that serum background effects increasedabsorbance values in the range of 0.3 OD units to approximately 0.5 ODunits depending on the serum sample being analyzed (data not shown). Itwas determined that the cause of the signal increase was HAMA effect(i.e. a reaction of human serum IgG with the mouse monoclonal antibodiesused in the assay) by testing human serum against RK22 coated oruncoated (control) HBX assay plates (FIG. 10). Plates without monoclonalantibody show no increase in signal suggesting the background increasewas not due to serum nonspecifically binding to the plate but wasdependent on the presence of monoclonal antibody.

Several attempts made to reduce background were unsuccessful, includingacid dissociation and the addition of excess IgG from various species toblock the binding of the human IgG to the murine IgGs (data not shown).The addition of a commercially available reagent, specifically designedto reduce HAMA assay interference Immunoglobulin Inhibiting Reagent (IIR) was successful when added to the following immunoassay. 1 ug/ml ofRK35 diluted in 0.1M Na Borate pH 8.5 was used as the capture reagent.Myo-029 was titrated onto the plate+/−serum+/−IIR and also 250 pg/mlGDF8 and incubated for 1.5 hours. Biotinylated RK22 was added at a1:10,000 dilution in THST assay buffer and incubated for 1.5 hours. 100μl of strepavidin-HRP diluted 1:20,000 in THST followed by developingthe reaction in TMB. As can be seen in FIG. 11, the HAMA background isreduced using the IIR reagent.

Example 6.3 Inhibition of GDF8 Binding with MyO-029 Antibody

MyO-029 (deposited on Oct. 2, 2002, at American Tissue CultureCollection (ATCC) under respective Deposit Designation Numbers PTA-4741)is a therapeutic antibody that has been used in clinical trials in aneffort to increase muscle strength in patients with muscular disorders.In the immunoassay to detect GDF8 in patients who have been administeredMyo-029, as described herein there has been shown to be cross-reactivityof assay antibody RK35 with the MYO-029 for binding to GDF-8. To verifythis cross-reactivity MYO-029 was coated onto assay plates. 1200 pg/mlGDF-8 was added to each well. Increasing concentrations of eitherbiotinylated RK22 or biotinylated RK35 were then added. As seen in FIG.12, unlabeled GDF-8 competes with biotinylated RK35 for binding toMYO-029 as no signal was generated in this configuration. No signal wasproduced with biotinylated RK35, which indicates cross reactivitybetween RK35 and Myo-029 for binding to GDF8.

Example 6.4 The Use of MyO-029 as a Competitor for GDF8

Assays were performed using both assay configurations (RK35/RK22 orRK22/RK35) and increasing amounts of MYO-029 therapeutic antibody with aconstant concentration of GDF-8 (250 pg/ml) spiked into assay buffer orinto 10% human serum. As seen in FIGS. 13A, RK22 was used as the captureantibody. GDF-8 at 250 pg/ml was incubated for 1.5 hours at roomtemperature with biotinylated RK35 at 150 ng/ml and increasingconcentrations of MYO-029 (0 to 20 ug/ml). Even at the highestconcentration of MYO-029 only approximately 30% inhibition was seen. Asshown in FIG. 13B, RK35 is used as the capture antibody. GDF-8 at 250pg/ml was incubated for 1.5 hours at room temperature with biotinylatedRK22 and increasing concentrations of MYO-029. Nearly 100% inhibition ofsignal was observed at concentrations of MYO-029≧5 ug/ml.

Example 6.5 Spike Recovery in Human Serum Using the RK35 Capture/RK22Detection Format

Three human serum samples were analyzed for GDF-8 recovery in spikerecovery experiments. Each sample was diluted to 600 pg/ml in 100% serumof mature GDF-8 and, serially diluted two fold to produce samples of600, 300, and 150 pg/ml of GDF-8. Each of these samples was analyzed inthe RK35 capture/RK22 detection format with or without the addition of20 μg/ml of MYO-029 see FIG. 14A. The reduction in signal due to theaddition of MYO-029 is calculated, see FIG. 14B. These values werecompared to a GDF-8 standard curve generated in THST assay buffer andobserved vs. expected values for GDF-8 are plotted in FIG. 15. Theobserved values are well below the expected values indicating thatstandard curves in buffer alone do not accurately quantitate GDF-8values found in serum samples.

Example 6.6 The Use of Serum Versus Assay Buffer for the Generation ofStandard Curves

Normal mouse serum and GDF-8 KO mouse serum was used in this study.These sera, along with normal human serum and THST buffer, were utilizedto generate standard curves using mature GDF-8 protein, see FIG. 15. Thedifference in slope between standard curves generated in serum vs.buffer explains why the observed vs. predicted values in spike/recoveryexperiments differ so drastically (FIG. 16) when buffer is the mediumused for generation of standard curves. The reduced slope of thestandard curve in serum compared to standard curve in buffer highlightsthe matrix effects of serum on the signal produced and suggests thatstandard curves should be generated in serum. KO mouse serum is notavailable in quantities necessary for assay development and normal mouseserum has high endogenous levels of GDF-8 that would interfere withprediction of GDF-8 in unknown serum samples. Normal human serum alsohas endogenous levels of GDF-8, albeit lower than normal mouse, thatalso adds an unwanted interference factor. A possible alternative wouldbe to deplete a pool of normal human serum of GDF-8 for use as an assaycalibration matrix.

Example 6.7 Inactivation/Dissociation of Myo-029 Antibody in Serum

Previous studies demonstrated that the presence of MYO-029 interferedwith the accurate quantitation of GDF-8 in the RK35/RK22 format bycross-reactivity with the epitope for the RK35 antibody. It washypothesized that if one could dissociate GDF-8/MYO-029 complexes in asample, it might be possible to develop an immunoassay that accuratelymeasures GDF-8 in the presence of MYO-029. A few possibilities exist fordissociation of antibodies from their antigens, including aciddissociation and heat denaturation. Based upon a report (Brown et al.,1990, Growth Factors 3, 35-43) that latent TGF-β could be irreversiblyactivated by heat treatment. A temperature gradient from 65° C. to 80°C. in THST assay buffer had no effect on the inhibition demonstrated byMYO-029 antibody in the GDF-8 assay. However, a pilot experiment withnormal unspiked serum, serum spiked with 400 pg/ml mature GDF-8, orserum spiked with latent GDF-8 was performed. In this experiment,MYO-029 antibody was spiked into 20% serum and heated to 80° C. forseven minutes (see FIG. 17). The results indicated that GDF-8 activityin the assay was retained at this temperature and that the MYO-029antibody was inactivated in serum heated to 80° C. This inactivation wasdemonstrated in_two ways: 1) serum samples heated in the presence ofMyo-029 had the same signal output as control heated samples with noMYO-029 addition, and 2) assay signal could again be reduced tobackground levels by the addition of fresh MYO-029 post sample heating.These results were observed in normal serum, serum spiked with matureGDF•8, and in serum spiked with latent GDF.8. Serum samples spiked withlatent GDF-8 demonstrate an increase in signal that seems dependent onlatent GDF-8 material and not on mature GDF.8. To determine a moreprecise duration for effective inactivation, a time course experimentwas conducted using normal serum spiked with 5 μg/ml MYO-029. Theresults indicated that the MYO-029 antibody is fully inactivated in asearly as three minutes at 80° C.

Example 6.8 GDF8 Depletion in Human Serum

In order to deplete human serum of endogenous GDF-8, an affinity columnwas made with 1 mg of MYO-029 antibody covalently immobilized toSepharose beads via cyanogen bromide activation. The column waspre-washed and small aliquots of human serum were passed over the columnand tested for GDF-8 activity in the GDF-8 ELISA assay. Additionally,serum was pre-heated to 65° C. and then cooled before being passed overthe column. Heated serum, when run in the GDF-8 assay, displays anincreased signal that may be due to immunological activation of latentGDF-8 in serum. FIG. 18 shows the signal increase in the GDF-8 assayupon heating and also the depletion of signal in the heated/depleted H/Dserum. Notably, there was no observed increase in signal in the H/Dserum when it was reheated to 80° C.

Example 6.9 Standard Curves in HID Serum

Using serum devoid of endogenous GDF-8 immunoreactivity standard curveswere generated under heated and unheated conditions using both matureGDF-8 and latent GDF-8. Mature GDF-8 was spiked into 100% serum andserially diluted from 2500 pg/ml to 40 pg/ml. Latent GDF-8 was used at aprotein concentration 20 times that of mature GDF8 ranging from 50,000pg/ml to 800 pg/ml (FIG. 19). The results of this experiment show asignificant increase in assay signal when latent GDF-8 is heated in 100%H/D serum to 80° C. Note that in FIG. 18 H/D serum heated to 80° C. didnot show the characteristic signal increase that is observed in normalserum. This lends credence to the hypothesis that the increase seen innormal serum is due to endogenous latent GDF-8. In contrast, the signalgenerated with mature GDF-8 does not increase upon heating and inrepeated experiments has a tendency to decrease with heating at 80° C.

Example 6.10 Analysis of Free and Total GDF8 in Eight Normal SerumSamples

Eight normal serum samples, previously frozen, thawed and assayed forfree and total GDF-8 using the room temperature assay (free) and the 80°C. heating step (total) to activate latent GDF-8. The standard curve forthis analysis was generated using H/D serum with known concentrations ofmature GDF-8 protein spiked in as the GDF-8 calibrator. The standardcurve ranged from 12,500 pg/ml to 188 pg/ml and a non-linear regressioncurve fit analysis with a correlation coefficient 0.999 (see FIG. 20)was used to calculate GDF-8 values in the eight unknown serum samples.As previously described, the change in optical density induced by theaddition of Myo-029 is directly proportional to endogenous amount ofGDF-8 in the sample. FIG. 21 is a presentation of the raw data that isused to determine the concentration of GDF-8. This figure illustratesthe free GDF-8 signal and its corresponding reduced signal by theaddition of MYO-029 antibody, as well as the total GDF-8 and itscorresponding reduced signal by the addition of MYO-029. FIG. 22presents the results of an assay reproducibility experiment. The freeand total GDF8 assay exhibits a high degree of reproducibility. This isespecially true for the free GDF-8 (room temperature) assay. Theincreased assay signal in the total (heated to 80° C.), assay from dayto day is due to an increase in heating time from five to ten minutes.The reduction in assay signal with increased time duration of sampleheating was observed repeatedly and may reflect denaturation of theantigen and loss of immunoreactivity.

Example 6.11 Heat Activated Endogenous GDF8 is Under Detected in thePresence of MYO-029

In the experiment shown in FIG. 23 human serum was incubated in thepresence or absence of MYO-029, then heat denatured at varioustemperatures prior to analysis by ELISA. Quantitation of GDF-8 levels intest samples was performed by interpolation from the assay results of astandard curve consisting of a dilution series of purified recombinantGDF-8 dimer of known concentration spiked into pooled human serumdepleted of GDF-8 by affinity chromatography. Maximum detection of GDF-8in the absence of MYO-029 occurred at approximately 60° C. The presenceof MYO-029 masked detection of GDF-8 in serum at low temperatures.Incubation at temperatures greater than 65° C. partially restoreddetection of GDF-8 in the presence of MYO-029. However, calculatedconcentrations of GDF-8 at temperatures greater than 65° C. showed thatthe assay substantially underestimated the total amount of GDF8 presentin the serum.

Example 6.12 GDF8 is Detected in the Presence of Myo-029 at Low pH

The suboptimal detection of GDF-8 when heated to temperatures necessaryfor dissociation of MYO-029 prompted evaluation of alternative methods.FIG. 24 demonstrates the ability of the acid dissociation ELISA todetect similar levels of GDF-8 in the absence and presence of MYO-029.In previous experiments the GDF-8/MYO-029 mixture was neutralized priorto binding to capture antibody. Under those conditions MYO-029 was ableto compete for binding with the capture antibody, RK35, uponneutralization. In FIG. 24 it is demonstrated that when the captureconditions are maintained at low acidic pH, RK35 is still capable ofbinding GDF-8 while MYO-029 is not, and therefore full recovery of GDF-8detection can be achieved in the presence or absence of therapeuticantibody.

Example 6.13 RK35 Binds GDF8 Under Acidic Conditions

To verify that RK35 (but not MYO-029) is capable of binding GDF-8 underacidic conditions, RK35 and MYO-029 were individually titrated intohuman serum and the analyte capture step was performed in acidicconditions. Increasing amounts of RK35 in solution was able to bind andcompete for GDF-8 binding to the plate-bound RK35, resulting indecreased detection of GDF-8 under acidic conditions. MYO-029 wasincapable of binding GDF-8 in solution approaching pH 3.0, regardless ofthe concentration of MYO-029 added, leaving the GDF-8 in solutionavailable to bind to the RK35 antibody coated in the ELISA well (seeFIG. 25). Thus, the ability of RK35 to bind GDF-8 under conditions inwhich MYO-029 cannot serves as a useful attribute that can be exploitedto measure GDF-8 levels in the presence of MYO-029. Acid dissociation ofMYO-029 from GDF-8 is effective even with escalating concentrations ofMYO-029. Data in FIG. 25 show that increasing the MYO-029 concentrationto 100 pg/ml did not diminish detection of GDF-8 using the aciddissociation protocol. This figure also shows the significant reductionin the detection of GDF-8 using the heat dissociation protocol, FIG. 26.

Acidification of human serum results in much greater estimates of GDF-8serum levels than previously reported. To demonstrate that the increasedserum estimates are the result of greater detection of GDF-8 and not dueto an acid induced artifact, serum from both a genetically engineeredknockout mouse and a naturally occurring GDF-8 knockout animal, theBelgian Blue cow, was analyzed. FIG. 27 shows that serum from both theknockout mouse and the Belgian Blue cow failed to produce a signal overplate background when measured at either neutral or acidic pH. Valuesreported here are in units of optical density and allow for comparativeevaluation however the lack of a calibrator curve results in theinability to determine the absolute quantity of GDF-8.

Example 6.14 Calibration Curve Fitting

To evaluate the inter-assay and intra-assay variability of theacid-dissociate method, aliquots of Belgian Blue serum spiked withrecombinant GDF-8 dimer were analyzed. Four stock solutions wereprepared independent of the calibration curve and were assayed intriplicate in five different locations of a 96 well plate.

A 4 or 5-parameter logistic model can be used to fit a calibration curvefor this assay. The arithmetic mean of the triplicates can be used asthe raw data for model fitting. Typically, variances at differentconcentration levels tend to be different. To obtain a calibration curvethat is accurate at both low and high concentrations, a weightednonlinear least squares method, or a variance-stabilizing transformationof the optical density data followed by a non-weighted nonlinear leastsquares method, should be employed.

FIGS. 28 A-C contrasts three different methods of calibration curvefitting for five GDF-8 ELISA plates in terms of their relative errors ofthe back-calculated concentrations for calibrating standards. If we take20% relative errors as acceptable, then both 4- and 5-parameter logisticmodel fitted on square root of optical density can be used.

Example 6.15 Precision and Accuracy of the Assay

To evaluate the -inter and -intra assay variability of theacid-dissociate method aliquots of Belgian Blue serum spiked withrecombinant GDF-8 dimer were analyzed. Four stock solutions wereprepared independent of the calibration curve and were assayed intriplicate in five different locations of a 96 well plate. FIG. 29 showsthe plate design of five plates that were processed on three differentdays. Concentrations of the spiked samples were calculated from acalibration curve fitted to each plate by a 5-parameter logistic model.Intra-plate and inter-plate coefficients of variation (CV) and relativeerrors (RE) are summarized in Tables 1 and 2. Both intra-plate andinter-plate precisions were well within 20% based on either method of CVcalculations.

TABLE 5 Intra-Plate Variability of Spiked Samples Average Nominal Numberof Average CV#I Average CV Relative Sample (netml) Plates (%) (%) (%)LLOQ 0.5 5 2.9 4.5 55.0 L-QC 2.0 5 2.4 3.2 37.1 M-QC 10.0 5 2.6 3.2 23.3H-QC 40.0 5 10.2 11.5 11.7 CV#1 = SD/Sample Mean CV#2 = SD/NominalConcentration

TABLE 6 Inter-Plate Variability of Spiked Samples Nominal SampleConcentration CV#I (%) CV#2 (%) Relative Error LLOQ 0.5 7.0 10.8 55.0L-QC 2.0 6.0 8.3 37.1 M-QC 10.0 7.2 8.9 23.3 H-QC 40.0 12.1 13.5 11.7CV#I = Inter-Plate SD/Sample Mean CV#2 = Inter-Plate SD/NominalConcentration

Example 7 Measurement of Myostatin Concentrations in Human Serum

Using the assay as described above, circulating concentrations ofmyostatin were measured and compared in healthy human sera. In one studythe sera of young and older men were evaluated for myostatin levels. Theeffects of testosterone treatment on circulating myostatin levels usingstored samples from a testosterone dose response study were alsoexamined. In that study, the details of which have been publishedpreviously (Bhasin S et al. J Clin Endocrinol Metab 2005; 90:678-88,Bhasin S et al. Am J Physiol Endocrinol Metab 2001; 281 :EI 172-81, andStorer T V V et al. J Clin Endocrinol Metab 2003; 88: 1478-85) theadministration of graded doses of testosterone to healthy young andolder men was associated with dose-dependent increases in skeletalmuscle mass and strength. In another study the sera of healthy andsurgically menopausal women were evaluated.

In the first study serum samples were derived from healthy young men,aged 18-35 years, and older men, aged 60-75 years, with normaltestosterone levels, who were participants in a testosterone doseresponse study as cited above. The study protocols were approved by theinstitutional review boards of Charles R. Drew University andHarbor-UCLA Research and Education Institute. Exclusion criteriaincluded a history of prostate cancer, PSA>4 ng/ml, a score of >7 on theAUA lower urinary tract symptoms questionnaire, a hematocrit >48%,severe sleep apnea, diabetes mellitus, congestive heart failure,myocardial infarct in the preceding six months, use of androgens in thepreceding year, or participation in moderate to intense exercisetraining regimens. After a four-week control period, participants wererandomly assigned to one of five treatment groups to receive monthlyinjections of a GnRH agonist (leuprolide depot, 7.5 mg; TAP, NorthChicago, Ill.) to suppress endogenous gonadotropin production. Theparticipants also received weekly intramuscular injections oftestosterone enanthate (TE, Delatestryl, 200 mg/ml; SavientPharmaceuticals, Inc., Iselin, N.J.) in one of five doses: 25 mg, 50 mg,125 mg, or 300 mg weekly.

Serum samples (or calibrator samples in Belgian Blue serum) were mixedwith acid dissociation buffer (0.2M Glycine-HCl pH 2.5) at a ratio of1:13.3. For non-dissociative assays, samples were mixed with THST buffer(50 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM Glycine, 0.05% Tween-20; pH8.0) instead of the Glycine-HCl buffer. Assay plates (Immulon 4 HBX#3855) were incubated with 2.0 mg/ml RK35 in coating buffer (100 mMSodium Borate, pH 9.1) overnight at 4° C., washed, and blocked with 200μl/well of SuperBlock-TBS (Pierce #37535). Diluted serum samples (100μL) were transferred to the assay plate, incubated at room temperaturefor 90 min, washed 4 times with THST and 100 μl biotinlylated RK-22secondary antibody (0.1 μg/ml) added to each well for 90 minutes at roomtemperature. Plates were washed four times with THST, and 100 μLStreptavidin-HRP (SouthernBiotech #7100-05) diluted 1:40,000 in THSTbuffer added for one hour at room temperature. Plates were washed againfour times with THST, and developed by addition of 100 μL of TMBsubstrate for 12 minutes at room temperature. 100 μl of 0.5M H₂S04 addedper well, and ELISA plates were read at OD 450 nm with wavelengthcorrection set at 540 nm.

Calibration curve range: A calibration curve was generated by plottingthe OD and corresponding concentration of each calibrator. A 5-parameterlogistic fit was used to fit the calibration curve. A calibration curveconsisting of two-fold dilutions of recombinant human mature myostatinin Belgian Blue serum extending from 73 pg/ml to 75,000 pg/mL wasprepared prior to each assay. The intra- and inter-assay imprecision ofthe read-back values for the eleven calibrators were determined from sixanalytical runs. The mean, SD, % CV, and % Bias of the extrapolatedconcentrations were calculated for each analytical run (to assessintra-assay imprecision) and for all analytical runs (to assessinter-assay imprecision). The lower and upper limits of quantitationwere defined as the lowest (LLQ) and highest (ULQ) calibratorconcentrations (respectively) that could be measured with an intraassay% CV and % Bias≦30%.

Preparation of Validation Samples: Three Sets of Validation SamplesCorresponding to low, mid-range, and high serum concentrations ofmyostatin were prepared using serum samples from healthy subjects withendogenous myostatin concentrations in the lower end and mid-range ofthe calibration curve, respectively. The high validation sample was aserum sample from a healthy subject spiked with recombinant myostatinprotein.

Intra- and Inter-Assay Imprecision: Intra- and inter-assay CVs weremeasured in six separate aliquots of each of the three validationsamples (low, mid, high) in five independent analytical runs. The QCanalytical run acceptance range (total assay variation mean+/−2SD) wasdetermined from myostatin concentrations measured in 23 separatealiquots of each of the three QC samples in 25 independent analyticalruns. One aliquot of each of the three QC samples (QC-low, QC-mid, andQC-high) was analyzed in each analytical run of samples. An analyticalrun was accepted if the measured concentrations of myostatin in two outof the three QC samples were within the established acceptance range.

Other assays: Serum total testosterone levels were measured by aspecific radioimmunoassay that has been validated previously againstliquid chromatography tandem massspectrometry (LC-MSI/MS). The intra-and inter-assay coefficients of variation for the total T assay were8.2% and 13.2%, respectively. Free T, separated from serum by anequilibrium dialysis procedure, was measured by a sensitiveradioimmunoassay that has a sensitivity of 0.22 pglml, and intra- andinter-assay coefficients of variation 4.2% and 12.3%, respectively(Sinha-Hikim et al. J Clin Endocrinol Metab 1998; 83:1312-8). Theradioimmunoassay and LC-MS/MS methods were compared by analyzing samplesprepared in charcoal stripped serum to which known amounts of T had beenadded. These measurements demonstrated a correlation of 0.99 between theradioimmunoassay and LC-MS/MS measurement. Serum sex hormone bindingglobulin (SHBG) levels were measured by an immunofluorometric assay thathas a sensitivity of 6.25 nmol/L. Body composition was assessed atbaseline and during week 20 by dual-energy X-ray absorptiometry (DEW,Hologic 4500, Waltham, Mass.). A body composition phantom was used tocalibrate the machine before each measurement.

Statistical Analyses: All outcome variables were evaluated fordistribution and homogeneity of variance; variables that did not meetthe assumptions of homogeneity of variance or normal distribution werelog-transformed. ANOVA was used to evaluate differences across dosegroups stratified by age, younger vs. older, at a single time point withSheffe's test to determine which groups differed significantly if adifference was identified by ANOVA. Changes within groups from baselineto treatment were evaluated with paired t-tests. Alpha was set at 0.05for determining statistical significance. Data are presented asmean+/−SEM or mean % change from baseline+/−SEM, unless otherwiseindicated in the figure legends.

Myostatin Assay Characteristics: Linear Range: The mean intra- andinter-assay imprecision was determined from six analytical runs for eachof the eleven calibrators (73-75,000 pg/mL) in the standard curve (Table7, below). The inter-assay CV for the 73 pg/mL calibrator was 36.4%,which exceeded the acceptable limit (<30%). Therefore, the LLQ of theassay was determined by the next calibrator point (147 pg/mL) at whichthe inter-assay CV and % Bias were 19.7% and 3.4%, respectively. Theinter-assay CV of 32.4% for the 75,000 pg/mL calibrator was also notwithin the acceptable limit (<30%) thereby defining the ULQ to the nextlowest calibrator point (37,500 pg/mL) at which the CV and % Bias were3.6% and 0.8%, respectively. The linear quantitative range of the assayextended from 143 pg/mL to 37,500 pg/mL in a biologically relevantmatrix.

TABLE 7 Intra and Inter-Assay Imprecision of the Myostatin CalibrationCurve Concentration (pg/mL) 75,000 37,500 18,750 9,375 4,688 2,344 1,172586 293 147 73 Intra-Assay CV Mean intra- 36.2 13.2 5 4.4 2.9 3.9 8.611.3 12.1 7.9 14.1 assay % CV # Analytical 2 6 6 6 6 6 6 6 6 6 6 RunsInter-Assay CV Analytical Run # Mean back-calculated concentrations fromindividual analytical runs 1 77,628 38,353 18,715 9,398 4,786 2,2741,168 541 271 122 154 2 48,648 39,596 18,532 9,393 4,906 2,284 1,012 608273 168 107 3 93,777 37,040 18,865 9,386 4,706 2,254 1,239 629 266 15955 4 47.567 35,705 18,875 9,354 4,674 2,407 1,123 605 280 117 106 553,495 37,477 18,862 9,224 4,889 2,316 1,070 611 261 149 109 6 45,91838,503 18,926 9,264 4,736 2,420 1,135 524 286 198 64 Mean 61,172 37,78918,796 9,336 4,782 2,326 1,124 586 273 152 99 (pg/mL) SD 19,837 1,348147 75 96 71 79 43 9 30 36 % CV 32.4 3.6 0.8 0.8 2 3.1 7 7.3 3.3 19.736.4 % Bias −18.4 0.8 0.2 −0.4 2.0 −0.8 −4.1 0.0 −6.8 3.4 35.6 N 6 6 6 66 6 6 6 6 6 6

Intra- and Inter-Assay Imprecision: The mean myostatin concentrations(pg/mL, +/−SD) in the low, mid, and high validation samples in fiveanalytical runs were 3739+/−146, 7615+/−125, and 18268+/−948,respectively. The measured intra-assay CV for the low, mid, and highvalidation samples (n=5 for each validation sample) was 4.1%, 4.7%, and7.2%, respectively, and the inter-assay CV was 3.9%, 1.6%, and 5.2%,respectively.

Assay Specificity: The mature myostatin protein has a high degree ofsequence conservation between mammalian species (4), allowing use of themyostatin assay on many non-human samples, including mouse, rat, dog,cow, and monkey. Serum samples from myostatin-deficient cattle (BelgianBlue) and from mice with an inactivating mutation in the myostatin gene(mstn KO) were assayed under dissociative acidic conditions, andcompared to normal animals of the same species (FIG. 29A). Serummyostatin was abundant in normal mice (˜120 ng/ml), while normal cowserum averaged approximately 40 ng/mL. In contrast, myostatin proteinwas undetectable in sera from both the Belgian Blue cattle and themyostatin KO mice, confirming the specificity of the assay for myostatinsince the mutations in these myostatin-null animals abolish thesynthesis of the protein.

Baseline Characteristics of Human Subjects: Fifty-two of the 61randomized young men and 51 of 60 randomized older men completed thetreatment phase. Sufficient serum for myostatin assays and bodycomposition data were available through week 20 for 50 young men and 48older men; these subjects were included in this secondary analysis andtheir baseline characteristics are shown in Table 8, below. Overall drugcompliance rate was greater than 99%.

The baseline total testosterone, free testosterone, percent freetestosterone, SHBG concentration, did not differ among the five dosegroups at baseline in either the young or older groups. However, oldermen had lower serum total and free testosterone, and higher SHBG thanyounger men. Body weight, body mass index, and percent fat mass weregreater in the older men than the younger men, while height was similarin both.

TABLE 8 Baseline Characterics of Evaluated Subjects Young Men (n = 50)Older Men (n = 48) Age (years) 26.5 ± 4.6 66.4 ± 4.7 Height (cm) 176.3 ±6.4  175.9 ± 5.7  Weight (kg)  75.1 ± 10.9  83.2 ± 11.7 BMI (kg/m2) 24.1± 3.0 26.9 ± 3.5 Lean body mass (kg) 57.6 ± 7.2 57.9 ± 6.3 Percent fatmass 18.0 ± 6.4 26.6 ± 5.4 Total testosterone level  578.4 ± 165.2 330.6± 96.1

Myostatin Levels in Young and Older Men: Serum myostatin levels werenormally distributed in both young and older men. Young men hadsignificantly higher myostatin levels than older men (8.0+/−0.3 vs.7.0+/−0.4 ng/mL, P=0.03) (FIG. 30A). Serum myostatin levels were notsignificantly correlated with lean body mass measured by DEXA in eitheryoung or older men (FIGS. 30B and 30C). Similarly, there was nosignificant correlation between myostatin levels and body weight, bodymass index, or serum testosterone levels at baseline (not shown).

Effects of Testosterone Administration on Myostatin Levels in Men: Serummyostatin levels at baseline did not differ significantly across thefive dose groups within either young or older men. Serum myostatinlevels were significantly higher on day 56 compared to baseline in bothyoung and older men (FIG. 31A). The changes in serum myostatinconcentrations did not differ significantly among the five dose groupseither within young or older men. Older men experienced a significantlygreater percent increase in myostatin levels than young men (FIG. 31B).The increases in myostatin levels during testosterone therapy were notsustained; thus, serum myostatin levels on day 140 were notsignificantly different from those at baseline.

The increments in myostatin levels above baseline were related tochanges in testosterone concentrations. Changes in myostatin levels frombaseline to day 56 were significantly positively correlated with changesin total (FIG. 32A) and free (FIG. 32C) testosterone concentrations inyoung men, but not in older men (FIGS. 32 B and 32D). As previouslyreported, testosterone treatment was associated with significant gainsin lean body mass; the changes in lean body mass were significantlycorrelated with testosterone dose and testosterone concentration, aspreviously described. However, changes in lean body mass were notsignificantly correlated with either absolute or percent change (FIGS.32E and 32F) in myostatin concentrations.

Myostatin Levels in Females: In another study, serum samples of healthy,young menstruating women, 19-21 years of age, and postmenopausal women,67-87 years of age, were purchased from BioServe, Beltsville, Md. Theseparticipants had consented to participate in an IRB-approved Bioservestudy. Surgically menopausal women were 18-55 years of age, who hadovarian surgery at least 6 months before enrollment and serum FSH>30U/L, BMI<35 kg/m², a normal PAP smear and mammogram in the preceding 12months, and who had provided a written informed consent approved by theBoston University IRB.

Serum myostatin levels in young women were not significantly differentfrom those in young men (FIG. 33). Myostatin levels in youngmenstruating women, surgically menopausal and naturally menopausal womendid not differ significantly.

1. An antagonist specific for Growth and Differentiation Factor 8(GDF8), wherein the antagonist is an anti-GDF8 antibody or antigenbinding protein that specifically binds to GDF8 and does notspecifically bind to BMP11 and wherein the antibody or antigen bindingprotein is selected from the group consisting of: polyclonal antibody; amonoclonal antibody; a monospecific antibody; polyspecific antibody;humanized antibody; a tetrameric antibody; a tetravalent antibody; amultispecific antibody; a single chain antibody; a domain-specificantibody; a single domain antibody; a domain-deleted antibody; a fusionprotein; an ScFc fusion protein; a single-chain antibody; chimericantibody; synthetic antibody; recombinant antibody; hybrid antibody;mutated antibody; CDR-grafted antibodies; an antibody fragmentcomprising an Fab; an F(ab′)2; an Fab′ fragment; an Fv fragment; asingle-chain Fv (ScFv) fragment; an Fd fragment; a dAb fragment; anantigen binding protein comprising diabodies; a CDR3 peptide; aconstrained FR3-CDR3-FR4 peptide; a nanobody; a bivalent nanobody; smallmodular immunopharmaceuticals (SMIPs); a shark variable IgNAR domain;and a minibody.
 2. The antagonist of claim 1, wherein the antibody orantigen binding protein comprises at least one complementaritydetermining region (CDR) comprising an amino acid sequence selected fromthe group consisting of: the amino acid sequence of SEQ ID NO:19, theamino acid sequence of SEQ ID NO:20, the amino acid sequence of SEQ IDNO:21, the amino acid sequence of SEQ ID NO:22, the amino acid sequenceof SEQ ID NO:23, the amino acid sequence of SEQ ID NO:24, the amino acidsequence of SEQ ID NO:25, the amino acid sequence of SEQ ID NO:26, theamino acid sequence of SEQ ID NO:27, the amino acid sequence of SEQ IDNO:28, the amino acid sequence of SEQ ID NO:29, the amino acid sequenceof SEQ ID NO:30.
 3. The antagonist of claim 2, wherein the antibody orantigen binding protein comprises a heavy chain, and wherein the heavychain comprises a first, second, and third complementarity determiningregion, wherein the first complementarity determining region comprisesan amino acid sequence selected from the group consisting of the aminoacid sequence of SEQ ID NO:19; and the amino acid sequence of SEQ IDNO:25 wherein the second complementarity determining region comprises anamino acid sequence selected from the group consisting of the amino acidsequence of SEQ ID NO:20; the amino acid sequence of SEQ ID NO:26 andwherein the third complementarity determining region comprises an aminoacid sequence selected from the group consisting of the amino acidsequence of SEQ ID NO:21; the amino acid sequence of SEQ ID NO:27. 4.The antagonist of claim 1, wherein the antibody or antigen bindingprotein comprises a heavy chain which comprises an amino acid sequenceselected from the group consisting of: the amino acid sequence of SEQ IDNO:14; and the amino acid sequence of SEQ ID NO:18.
 5. The antagonist ofclaim 2, wherein the antibody or antigen binding protein comprises alight chain, and wherein the light chain comprises a first, second, andthird complementarity determining region, wherein the firstcomplementarity determining region comprises an amino acid sequenceselected from the group consisting of the amino acid sequence of SEQ IDNO:22; and the amino acid sequence of SEQ ID NO:28 wherein the secondcomplementarity determining region comprises an amino acid sequenceselected from the group consisting of the amino acid sequence of SEQ IDNO:23; and the amino acid sequence of SEQ ID NO:29 and wherein the thirdcomplementarity determining region comprises an amino acid sequenceselected from the group consisting of the amino acid sequence of SEQ IDNO:24; and the amino acid sequence of SEQ ID NO:30.
 6. The antagonist ofclaim 1, wherein antibody or antigen binding protein comprises a lightchain which comprises an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO:16; and the aminoacid sequence of SEQ ID NO:17.
 7. The antagonist of claim 1, wherein theantibody or antigen binding protein comprises a light chain whichcomprises an amino acid sequence of SEQ ID NO:16, and wherein theantibody further comprises a heavy chain comprising the amino acidsequence of SEQ ID NO:14.
 8. The antagonist of claim 1, wherein theantibody or antigen binding protein comprises a light chain whichcomprises the amino acid sequence of SEQ ID NO:17, and wherein theantibody further comprises a heavy chain which comprises the amino acidsequence of SEQ ID NO:18.
 9. A polynucleotide that encodes amino acidscomprising the GDF8 antagonist as in either of claims 7 or
 8. 10. A hostcell comprising a polynucleotide as in claim 9, wherein thepolynucleotide is operably linked to a regulatory sequence.
 11. A vectorcomprising a polynucleotide as in claim
 9. 12. A host cell comprisingthe vector as in claim
 11. 13. A method for producing a GDF8 antagonistcomprising culturing a host cell comprising a polynucleotide as in claim12, and isolating the GDF8 antagonist expressed by the host cell. 14.The isolated GDF8 antagonist produced by the method of claim
 13. 15. Anassay to detect the presence of GDF8 in a sample from a subject, theassay comprising the following steps: (a) combining (i) the sample; (ii)a capture reagent that specifically binds GDF8; (iii) a detectionreagent that specifically binds GDF8; and (b) detecting whether or notspecific binding occurs between the capture reagent and GDF8 whereindetection of specific binding indicates the presence of GDF8 in thesample.
 16. The assay of claim 15 which further comprises combining thesample with an acidic buffer prior to the combination in (a).
 17. Theassay according to claim 16 wherein the pH of the acidic buffer isbetween about pH 1.0 and pH 6.0.
 18. The assay according to claim 16wherein the pH of the acidic buffer is about pH 2.5.
 19. The assay ofclaim 15 wherein the capture reagent is selected from the groupconsisting of an anti-GDF8 antibody or antigen binding protein; a GDF8binding protein; and a GDF8 binding domain.
 20. The assay of claim 19wherein the capture reagent is an anti-GDF8 antibody or antigen bindingprotein and is selected from the group of consisting of: RK35 and RK22.21. The assay of claim 20 wherein the capture reagent is RK35.
 22. Theassay of claim 20 wherein the capture reagent is RK22.
 23. The assay ofclaim 15 wherein the detection reagent is selected from the groupconsisting of an anti-GDF8 antibody or antigen binding protein; a GDF8binding protein; and a GDF8 binding domain.
 24. The assay of claim 23wherein the detection reagent is an anti-GDF8 antibody or antigenbinding protein and is selected from the group consisting of RK35 andRK22.
 25. The assay of claim 24 wherein the detection reagent is RK35.26. The assay of claim 24 wherein the detection reagent is RK22.
 27. Anassay for quantitating GDF8 in a sample from a subject, the assaycomprising the following steps: (a) combining (i) the sample; (ii) acapture reagent that specifically binds GDF8; (iii) a detection reagentthat specifically binds GDF8; and (b) detecting whether or not aspecific binding occurs between the capture reagent and GDF8; and (c)quantitating the level of GDF8; wherein detection of specific bindingthat indicates the presence of GDF8 in the sample can be quantitated.28. The assay according to claim 27 which further comprises combiningthe sample with an acidic buffer prior to the combination in (a). 29.The assay according to claim 28 wherein the pH of the acidic buffer isbetween about pH 1.0 and pH 6.0.
 30. The assay according to claim 28wherein the pH of the acidic buffer is about pH 2.5.
 31. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a GDF8 antagonist wherein the antagonist is an anti-GDF8antibody or antigen binding protein that specifically binds to GDF8 anddoes not specifically bind to BMP11.
 32. A pharmaceutical composition asin claim 31 wherein the anti-GDF8 antibody or antigen binding proteinthat specifically binds with GDF8 and does not specifically bind BMP11comprises a light chain comprising the amino acid sequence of SEQ IDNO:16, and wherein the antibody further comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:14.
 33. A pharmaceuticalcomposition as in claim 31 wherein the anti-GDF8 antibody or antigenbinding protein that specifically binds with GDF8 and does notspecifically bind BMP11 comprises a light chain comprising the aminoacid sequence of SEQ ID NO:18, and wherein the antibody furthercomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:17.
 34. A pharmaceutical composition as in claim 31 wherein theanti-GDF8 antibody that specifically binds with GDF8 and does not bindBMP11 comprises at least one complementarity determining region (CDR)comprising an amino acid sequence selected from the group consisting ofthe amino acid sequence of SEQ ID NO:19, the amino acid sequence of SEQID NO:20, the amino acid sequence of SEQ ID NO:21, the amino acidsequence of SEQ ID NO:22, the amino acid sequence of SEQ ID NO:23, theamino acid sequence of SEQ ID NO:24, the amino acid sequence of SEQ IDNO:25, the amino acid sequence of SEQ ID NO:26, the amino acid sequenceof SEQ ID NO:27, the amino acid sequence of SEQ ID NO:28, the amino acidsequence of SEQ ID NO:29, the amino acid sequence of SEQ ID NO:30.
 35. Amethod of treating a GDF8-associated disorder in a mammalian patientcomprising administering to the patient a therapeutically effectiveamount of an antagonist specific for GDF8 that has little to notoxicity.
 36. The method of claim 35, wherein the GDF8 antagonistis ananti-GDF8 antibody or antigen binding protein that specifically binds toGDF8 and does not specifically bind to BMP11.
 37. A method as in claim36 wherein the GDF8 antagonist is an anti-GDF8 antibody or antigenbinding protein that specifically binds with GDF8 and does notspecifically bind to BMP11 comprises a light chain comprising the aminoacid sequence of SEQ ID NO:16, and wherein the antibody furthercomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:14.
 38. A method as in claim 36 wherein the GDF8 antagonist is ananti-GDF8 antibody or antigen binding protein that specifically bindswith GDF8 and does not specifically bind to BMP11 comprises a lightchain comprising the amino acid sequence of SEQ ID NO:18, and whereinthe antibody further comprises a heavy chain comprising the amino acidsequence of SEQ ID NO:17.
 39. A method as in claim 36 wherein theanti-GDF8 antibody or antigen binding protein that specifically bindswith GDF8 and does not specifically bind to BMP11 comprises at least onecomplementarity determining region (CDR) comprising an amino acidsequence selected from the group consisting of the amino acid sequenceof SEQ ID NO:19, the amino acid sequence of SEQ ID NO:20, the amino acidsequence of SEQ ID NO:21, the amino acid sequence of SEQ ID NO:22, theamino acid sequence of SEQ ID NO:23, the amino acid sequence of SEQ IDNO:24, the amino acid sequence of SEQ ID NO:25, the amino acid sequenceof SEQ ID NO:26, the amino acid sequence of SEQ ID NO:27, the amino acidsequence of SEQ ID NO:28, the amino acid sequence of SEQ ID NO:29, theamino acid sequence of SEQ ID NO:30.
 40. A method of diagnosing,determining a prognosis for, or detecting whether a subject is afflictedwith a GDF8-associated disorder comprising the steps of: (a) obtaining afirst sample from the subject; (b) combining a first sample with theantagonist as in claim 9; (c) detecting the presence of GDF8 in thefirst sample; (d) quantitating the level of GDF8 in the first sample;(e) obtaining a second sample from a subject not afflicted with theGDF8-associated disorder; (f) combining the second sample with theantagonist; (g) detecting the level of GDF8 in the second sample; (h)quantitating the level of GDF9 in the second sample and (i) comparingthe levels of GDF8 in the first and second samples, wherein an increase,decrease, or similarity in the level of GDF8 in the first samplecompared to the second sample indicates whether the subject is afflictedwith a GDF8-associated disorder.
 41. The use of a pharmaceuticalcomposition in the preparation of a medicament for treating aGDF8-associated disorder in a mammalian patient wherein thepharmaceutical composition comprises a pharmaceutically acceptablecarrier and a GDF8 antagonist wherein the GDF8 antagonist is ananti-GDF8 antibody or antigen binding protein that specifically binds toGDF8 and does not specifically bind to BMP11.
 42. A kit for detectingthe presence of or quantitating GDF8 in a sample from a subject, the kitcomprising a capture reagent that specifically binds GDF8 and adetection reagent that specifically binds GDF8 wherein detection ofspecific binding of GDF8 to the capture and detection reagents indicatethe presence of GDF8 in the sample.
 43. The kit as in claim 78 whichfurther comprises an acidic buffer.
 44. An antibody specific for Growthand Differentiation Factor 8 (GDF8), wherein the anti-GDF8 antibody orantigen binding protein that specifically binds to GDF8 and does notspecifically bind to BMP11 and wherein the antibody or antigen bindingprotein is selected from the group consisting of: polyclonal antibody; amonoclonal antibody; a monospecific antibody; polyspecific antibody;humanized antibody; a tetrameric antibody; a tetravalent antibody; amultispecific antibody; a single chain antibody; a domain-specificantibody; a single domain antibody; a domain-deleted antibody; a fusionprotein; an ScFc fusion protein; a single-chain antibody; chimericantibody; synthetic antibody; recombinant antibody; hybrid antibody;mutated antibody; CDR-grafted antibodies; an antibody fragmentcomprising an Fab; an F(ab′)2; an Fab′ fragment; an Fv fragment; asingle-chain Fv (ScFv) fragment; an Fd fragment; a dAb fragment; anantigen binding protein comprising diabodies; a CDR3 peptide; aconstrained FR3-CDR3-FR4 peptide; a nanobody; a bivalent nanobody; smallmodular immunopharmaceuticals (SMIPs); a shark variable IgNAR domain;and a minibody.
 45. The antibody of claim 44 wherein the antibody orantigen binding protein is a monoclonal antibody.
 46. The antibody ofclaim 45 wherein the antibody or antigen binding protein comprises alight chain which comprises an amino acid sequence of SEQ ID NO: 16, andwherein the antibody further comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:14.
 47. The antibody of claim 44wherein the antibody or antigen binding protein is a humanized antibody.48. The antibody of claim 47 wherein the antibody or antigen bindingprotein comprises a light chain which comprises the amino acid sequenceof SEQ ID NO:17, and wherein the antibody further comprises a heavychain which comprises the amino acid sequence of SEQ ID NO:18.